Detergent compositions and uses thereof

ABSTRACT

The present invention relates to polypeptides having hexosaminidase activity, and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application ofPCT/EP2017/060265 filed Apr. 28, 2017, which claims priority or thebenefit under 35 U.S.C. 119 of Denmark application PA 2016 00262 filedApr. 29, 2016, the contents of which are fully incorporated herein byreference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to polypeptides having hexosaminidaseactivity, polynucleotides encoding the polypeptides and catalyticdomains belonging to the Glycoside Hydrolase family 20 (GH20,www.cazy.org). The invention further relates to compositions comprisingsuch polypeptides in particular cleaning compositions, use ofpolypeptides having hexosaminidase activity in cleaning processes and/orfor deep cleaning, methods for deep cleaning. The invention furtherrelates to nucleic acid constructs, vectors, and host cells comprisingthe polynucleotides as well as methods of producing and using thepolypeptides and catalytic domains.

Description of the Related Art

Polypeptides having hexosaminidase activity include Dispersins such asDispersin B (DspB), which are described as β-N-acetylglucosamininidasesbelonging to the Glycoside Hydrolase 20 family. WO04061117 A2 (KaneBiotech INC) describe use of compositions comprising DspB for reducingand preventing biofilm caused by poly-N-acetylglucosamine-producingbacteria and

Kane et al. describes the use of compositions comprising dispersins forreducing biofilm on medical devises and for wound care. Biofilm may alsobe present on laundry items, such as fabrics, other hard surfaces, suchas dish wash utensils, dish washers and washing machines where they maycause malodor, which is difficult to remove even after wash. Theapplication WO9850512 (Procter and Gamble) disclose laundry or cleaningproducts comprising one or more hexosaminidase enzymes. The presentinvention provides suitable enzymes for use in detergents and for deepcleaning of items such as laundry and cleaning process.

SUMMARY OF THE INVENTION

The present invention provides polypeptides belonging to theTerribacillus clade of hexosaminidases from the Glycoside Hydrolase 20family. The Terribacillus clade is shown in FIGS. 1 and 3. Thepolypeptides of the invention have hexosaminidase activity. Theinvention further provides detergent compositions comprisingpolypeptides having hexosaminidase activity and the use of polypeptideshaving hexosaminidase activity for cleaning processes. The polypeptidesof the present invention having hexosaminidase activity have beneficialproperties such as removal and/or reduction of biofilm relatedcomponents such as EPS and/or PNAG in cleaning processes. Includinglaundry and dish wash. The polypeptides of the present invention belongto the Terribacillus clade, which are homologous sequences of aparticular clade of the Glycoside Hydrolase 20 family. Accordingly, thepresent invention relates to polypeptides having hexosaminidase activityselected from the group consisting of:

-   -   (a) a polypeptide having at least 80% sequence identity to the        mature polypeptide of SEQ ID NO 2, 4, 6, 13 or 15;    -   (b) a variant of the mature polypeptide of SEQ ID NO 2, 4, 6, 13        or 15 comprising a substitution, deletion, and/or insertion at        one or more (e.g., several) positions; and    -   (c) a fragment of the polypeptide of (a) or (b) that has        hexosaminidase activity.

The present invention also relates to polypeptides comprising acatalytic domain belonging to the Glycoside Hydrolase family 20 (GH20,www.cazy.org) and having at least 60% sequence identity to amino acids 1to 324 of SEQ ID NO 2; at least 60% sequence identity to amino acids 1to 324 of SEQ ID NO 4, at least 60% sequence identity to amino acids 1to 324 of SEQ ID NO 6, at least 60% sequence identity to amino acids 1to 324 of SEQ ID NO 13 or at least 60% sequence identity to amino acids1 to 324 of SEQ ID NO 15. The present invention also relates to cleaningmethods using the polypeptides of the present invention and to the usein cleaning processes. The invention further relates to a method forcleaning or laundering an item comprising the steps of:

-   -   a. exposing an item to a wash liquor comprising a polypeptide        having hexosaminidase activity selected from the group        consisting of the polypeptides of SEQ ID NO 7, 8, 9, 10 and 11        or a polypeptide having at least 60% sequence identity hereto or        a detergent composition comprising the polypeptides;    -   b. Completing at least one wash cycle; and    -   c. Optionally rinsing the item,    -   wherein the item is a textile.

In addition is claimed the use of a polypeptide having hexosaminidaseactivity selected from the group consisting of the polypeptides of SEQID NO 7, 8, 9, 10, 11 or a polypeptide having at least 60% sequenceidentity for deep cleaning of an item.

The invention further relates to a composition comprising at least 0.001ppm polypeptide having hexosaminidase activity, wherein the polypeptideis selected from the group consisting of polypeptides having at least60% sequence identity to the mature polypeptide shown in SEQ ID NO 7, 8,9, 10 and 11; and at least one adjunct ingredient. The invention furtherrelates to the use of a composition of the invention for deep-cleaningof an item, wherein the item is a textile.

The invention further relates to a method for laundering an itemcomprising: a) expose an item to a wash liquor comprising a polypeptideselected from the group consisting of polypeptides shown in SEQ ID NO 7,SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11 or a polypeptidehaving at least 60% sequence identity hereto or expose of an item to adetergent composition according to the invention; b) completing at leastone wash cycle; and optionally rinsing the item, wherein the item is atextile.

The invention further relates to the use of a polypeptide or acomposition of the invention,

-   -   (i) for preventing, reducing or removing stickiness of the item;    -   (ii) for pretreating stains on the item;    -   (iii) for preventing, reducing or removing redeposition of soil        during a wash cycle;    -   (iv) for preventing, reducing or removing adherence of soil to        the item;    -   (v) for maintaining or improving whiteness of the item;    -   (vi) for preventing, reducing or removal malodor from the item,        wherein the item is a textile.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Phylogenic tree showing the Terribacillus clade.

FIG. 2 An alignment of the polypeptides of the invention

FIG. 3 A phylogenetic tree of the polypeptides of the invention

OVERVIEW OF SEQUENCES OF THE TERRIBACILLUS CLADE

SEQ ID NO 1 is the DNA encoding the full-length polypeptide fromTerribacillus saccharophilus

SEQ ID NO 2 is the polypeptide derived from SEQ ID NO 1

SEQ ID NO 3 is the DNA encoding the full-length polypeptide fromTerribacillus goriensis

SEQ ID NO 4 is the polypeptide derived from SEQ ID NO 3

SEQ ID NO 5 is the DNA encoding the full-length polypeptide fromTerribacillus saccharophilus

SEQ ID NO 6 is the polypeptide derived from SEQ ID NO 5

SEQ ID NO 7 is the mature polypeptide of SEQ ID NO 2

SEQ ID NO 8 is the mature polypeptide of SEQ ID NO 4

SEQ ID NO 9 is the mature polypeptide of SEQ ID NO 6

SEQ ID NO 10 is the mature polypeptide of SEQ ID NO 13

SEQ ID NO 11 is the mature polypeptide of SEQ ID NO 15

SEQ ID NO 12 is the DNA encoding the full-length polypeptide fromTerribacillus saccharophilus

SEQ ID NO 13 is the polypeptide derived from SEQ ID NO 12

SEQ ID NO 14 is the DNA encoding the full-length polypeptide fromTerribacillus saccharophilus

SEQ ID NO 15 is the polypeptide derived from SEQ ID NO 14

SEQ ID NO 16 is the Bacillus clausii secretion signal

SEQ ID NO 17 is a His-tag sequence

SEQ ID NO 18 is the polypeptide motif GXDE

SEQ ID NO 19 is the polypeptide motif[EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN]

SEQ ID NO 20 is the polypeptide motif [VIM][LIV]G[GAV]DE[VI][PSA]

SEQ ID NO 21 is the polypeptide motif WND[SQR][IVL][TLVM]

SEQ ID NO 22 is the polypeptide motif QSTL

SEQ ID NO 23 is the polypeptide motif NKFFY

SEQ ID NO 24 is the polypeptide motif NLD[DR]S

Definitions

Dispersin: The term “dispersin” and the abbreviation “Dsp” means apolypeptide having hexosaminidase activity, EC 3.2.1.—that catalyzes thehydrolysis of β-1,6-glycosidic linkages of N-acetyl-glucosamine polymers(poly-N-acetylglucosamine) found e.g. in biofilm.

Hexosaminidase: The term “hexosaminidases” means a polypeptide havinghexosaminidase activity (hexosaminidases), and includes EC 3.2.1.e.g.that catalyzes the hydrolysis of of N-acetyl-D-hexosamine orN-acetyl-glucosamine polymers found e.g. in biofilm. The term includesdispersins and includes polypeptides having N-acetylglucosaminidaseactivity and β-N-acetylglucosamininidase activity. The term “polypeptidehaving hexosaminidase activity” may be used interchangeably with theterm hexosaminidases and similar the term “polypeptide havingβ-N-acetylglucosaminidase activity” may be used interchangeably with theterm β-N-acetylglucosamininidases. For the purposes of the presentinvention, hexosaminidase activity is determined according to theprocedure described in Assay 1 or 2. In one aspect, the polypeptides ofthe present invention have at least 20%, e.g., at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, or at least 100% of the hexosaminidase activity of the maturepolypeptide of SEQ ID NO 2. In one aspect, the polypeptides of thepresent invention have at least 20%, e.g., at least 40%, at least 50%,at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, orat least 100% of the hexosaminidase activity of the mature polypeptideof SEQ ID NO 4. In one aspect, the polypeptides of the present inventionhave at least 20%, e.g., at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, or at least 100% ofthe hexosaminidase activity of the mature polypeptide of SEQ ID NO 6. Inone aspect, the polypeptides of the present invention have at least 20%,e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95%, or at least 100% of the hexosaminidaseactivity of the mature polypeptide of SEQ ID NO 13. In one aspect, thepolypeptides of the present invention have at least 20%, e.g., at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, or at least 100% of the hexosaminidase activity ofthe mature polypeptide of SEQ ID NO 15.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Biofilm: A biofilm may be produced by any group of microorganisms inwhich cells stick to each other or stick to a surface, such as atextile, dishware or hard surface or another kind of surface. Theseadherent cells are frequently embedded within a self-produced matrix ofextracellular polymeric substance (EPS). Biofilm EPS is a polymericconglomeration generally composed of extracellular DNA, proteins, andpolysaccharides. Biofilms may form on living or non-living surfaces. Themicrobial cells growing in a biofilm are physiologically distinct fromplanktonic cells of the same organism, which, by contrast, aresingle-cells that may float or swim in a liquid medium. Bacteria livingin a biofilm usually have significantly different properties fromplanktonic bacteria of the same species, as the dense and protectedenvironment of the film allows them to cooperate and interact in variousways. One benefit of this environment for the microorganisms isincreased resistance to detergents and antibiotics, as the denseextracellular matrix and the outer layer of cells protect the interiorof the community. On laundry and hard surfaces biofilm producingbacteria can be found among the following species: Acinetobacter sp.,Aeromicrobium sp., Brevundimonas sp., Microbacterium sp Micrococcusluteus, Pseudomonas sp., Streptococcus sp., Streptococcus dysgalactiae,Staphylococcus epidermidis, Staphylococcus aureus, Staphylococcuspneumoniae, Stenotrophomonas sp., Enterobacter sp., Xanthomonas sp.,Yersinia sp., Klebsiella sp., Burkholderia sp., Stenotrophomonas sp.,Variovorax sp., Escherichia sp., Ralstonia sp., Achromobacter sp.,Luteibacter sp., Citrobacter sp., Xanthomonadaceae sp., Halomonas sp.,Bordetella sp., Lysobacter sp., Serratia sp., Escherichia sp.,Aggregatibacter sp., Listeria monocytogenes, Clostridium difficile,Mycobacterium sp., Neisseria gonorrheae, H. influenzae, Haemophilusducreyi, Helicobacter pylori, Campylobacter jejuni and Enterococcusfaecalis as well as the fungi Candida albicans, Aspergillus flavus,Fusarium solani, and Cryptococcus neoformans. In one aspect, the biofilmcomponent e.g. poly-N-acetylglucosamine comprising strain isBrevundimonas sp. In one aspect, the biofilm component e.g.poly-N-acetylglucosamine comprising strain is Pseudomonas alcaliphila orPseudomonas fluorescens. In one aspect, the biofilm component e.g.poly-N-acetylglucosamine comprising strain is Staphylococcus aureus.

Catalytic domain: The term “catalytic domain” means the region of anenzyme containing the catalytic machinery of the enzyme.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Clade: a group of polypeptides clustered together based on homologousfeatures traced to a common ancestor. Polypeptide clades can bevisualized as phylogenetic trees and a clade is a group of polypeptidesthat consists of a common ancestor and all its lineal descendants (FIG.1). The polypeptides of the invention e.g. all belong to theTerribacillus clade, which is illustrated as a phylogenetic tree inFIGS. 1 and 3. The Terribacillus clade or clade of Terribacillus is agroup of enzymes all related to the same ancestor and share commonproperties. Polypeptides forming a group within the clade (a subclade)of the phylogenetic tree can also share common properties and are moreclosely related than other polypeptides in the clade.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG, or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding a maturepolypeptide of the present invention. Each control sequence may benative (i.e., from the same gene) or foreign (i.e., from a differentgene) to the polynucleotide encoding the polypeptide or native orforeign to each other. Such control sequences include, but are notlimited to, a leader, polyadenylation sequence, propeptide sequence,promoter, signal peptide sequence, and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe polynucleotide encoding a polypeptide.

Deep cleaning: By the term “deep cleaning” is meant reduction or removalof components of biofilm, such as EPS or parts hereof, polysaccharides,PNAG (poly-N-acetylglucosamine), proteins, DNA, soil or other componentspresent in the biofilm.

Detergent adjunct ingredient: The detergent adjunct ingredient isdifferent to the hexosaminidase of this invention. The precise nature ofthese additional adjunct components, and levels of incorporationthereof, will depend on the physical form of the composition and thenature of the operation for which it is to be used. Suitable adjunctmaterials include, but are not limited to the components described belowsuch as surfactants, builders, flocculating aid, chelating agents, dyetransfer inhibitors, enzymes, enzyme stabilizers, enzyme inhibitors,catalytic materials, bleach activators, hydrogen peroxide, sources ofhydrogen peroxide, preformed peracids, polymeric agents, clay soilremoval/anti-redeposition agents, brighteners, suds suppressors, dyes,perfumes, structure elasticizing agents, fabric softeners, carriers,hydrotropes, builders and co-builders, fabric huing agents, anti-foamingagents, dispersants, processing aids, and/or pigments.

Detergent Composition: The term “detergent composition” refers tocompositions that find use in the removal of undesired compounds fromitems to be cleaned, such as textiles. The detergent composition may beused to e.g. clean textiles for both household cleaning and industrialcleaning. The terms encompass any materials/compounds selected for thecleaning composition desired and the form of the product (e.g., liquid,gel, powder, granulate, paste, or spray compositions) and includes, butis not limited to, detergent compositions (e.g., liquid and/or solidlaundry detergents and fine fabric detergents; fabric fresheners; fabricsofteners; and textile and laundry pre-spotters/pretreatment). Inaddition to containing the polypeptide of the invention, the compositionmay contain one or more additional enzymes (such as proteases, amylases,lipases, cutinases, cellulases, endoglucanases, xyloglucanases,pectinases, pectin lyases, xanthanases, peroxidases, haloperoxygenases,catalases and mannanases, or any mixture thereof), and/or detergentadjunct ingredients such as surfactants, builders, chelators orchelating agents, bleach system or bleach components, polymers, fabricconditioners, foam boosters, suds suppressors, dyes, perfume, tannishinhibitors, optical brighteners, bactericides, fungicides, soilsuspending agents, anti-corrosion agents, enzyme inhibitors orstabilizers, enzyme activators, transferase(s), hydrolytic enzymes,oxido reductases, bluing agents and fluorescent dyes, antioxidants, andsolubilizers.

Enzyme Detergency benefit: The term “enzyme detergency benefit” isdefined herein as the advantageous effect an enzyme may add to adetergent compared to the same detergent without the enzyme. Importantdetergency benefits which can be provided by enzymes are stain removalwith no or very little visible soils after washing and/or cleaning,prevention or reduction of redeposition of soils released in the washingprocess (an effect that also is termed anti-redeposition), restoringfully or partly the whiteness of textiles which originally were whitebut after repeated use and wash have obtained a greyish or yellowishappearance (an effect that also is termed whitening). Textile carebenefits, which are not directly related to catalytic stain removal orprevention of redeposition of soils, are also important for enzymedetergency benefits. Examples of such textile care benefits areprevention or reduction of dye transfer from one fabric to anotherfabric or another part of the same fabric (an effect that is also termeddye transfer inhibition or anti-backstaining), removal of protruding orbroken fibers from a fabric surface to decrease pilling tendencies orremove already existing pills or fuzz (an effect that also is termedanti-pilling), improvement of the fabric-softness, colour clarificationof the fabric and removal of particulate soils which are trapped in thefibers of the fabric or garment. Enzymatic bleaching is a further enzymedetergency benefit where the catalytic activity generally is used tocatalyze the formation of bleaching components such as hydrogen peroxideor other peroxides.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to control sequences that provide forits expression.

Fragment: The term “fragment” means a polypeptide or a catalytic domainhaving one or more (e.g., several) amino acids absent from the aminoand/or carboxyl terminus of a mature polypeptide or domain; wherein thefragment has hexosaminidase activity. In one aspect, a fragment containsat least 300 amino acid residues (e.g., amino acids 1 to 300 of SEQ IDNO 2), at least 305 amino acid residues (e.g., amino acids 1 to 305 ofSEQ ID NO 2), at least 310 amino acid residues (e.g., amino acids 1 to310 of SEQ ID NO 2), at least 315 amino acid residues (e.g., amino acids1 to 315 of SEQ ID NO 2), or at least 320 amino acid residues (e.g.,amino acids 1 to 320 of SEQ ID NO 2). In one aspect, a fragment containsat least 300 amino acid residues (e.g., amino acids 1 to 300 of SEQ IDNO 4), at least 305 amino acid residues (e.g., amino acids 1 to 305 ofSEQ ID NO 4), at least 310 amino acid residues (e.g., amino acids 1 to310 of SEQ ID NO 4), at least 315 amino acid residues (e.g., amino acids1 to 315 of SEQ ID NO 4), or at least 320 amino acid residues (e.g.,amino acids 1 to 320 of SEQ ID NO 4). In one aspect, a fragment containsat least 300 amino acid residues (e.g., amino acids 1 to 300 of SEQ IDNO 6), at least 305 amino acid residues (e.g., amino acids 1 to 305 ofSEQ ID NO 6), at least 310 amino acid residues (e.g., amino acids 1 to310 of SEQ ID NO 6), at least 315 amino acid residues (e.g., amino acids1 to 315 of SEQ ID NO 6), or at least 320 amino acid residues (e.g.,amino acids 1 to 320 of SEQ ID NO 6). In one aspect, a fragment containsat least 300 amino acid residues (e.g., amino acids 1 to 300 of SEQ IDNO 13), at least 305 amino acid residues (e.g., amino acids 1 to 305 ofSEQ ID NO 13), at least 310 amino acid residues (e.g., amino acids 1 to310 of SEQ ID NO 13), at least 315 amino acid residues (e.g., aminoacids 1 to 315 of SEQ ID NO 13), or at least 320 amino acid residues(e.g., amino acids 1 to 320 of SEQ ID NO 13). In one aspect, a fragmentcontains at least 300 amino acid residues (e.g., amino acids 1 to 300 ofSEQ ID NO 15), at least 305 amino acid residues (e.g., amino acids 1 to305 of SEQ ID NO 15), at least 310 amino acid residues (e.g., aminoacids 1 to 310 of SEQ ID NO 15), at least 315 amino acid residues (e.g.,amino acids 1 to 315 of SEQ ID NO 15), or at least 320 amino acidresidues (e.g., amino acids 1 to 320 of SEQ ID NO 15).

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., recombinantproduction in a host cell; multiple copies of a gene encoding thesubstance; and use of a stronger promoter than the promoter naturallyassociated with the gene encoding the substance). An isolated substancemay be present in a fermentation broth sample; e.g. a host cell may begenetically modified to express the polypeptide of the invention. Thefermentation broth from that host cell will comprise the isolatedpolypeptide.

Improved wash performance: The term “improved wash performance” isdefined herein as an enzyme displaying an increased wash performance ina detergent composition relative to the wash performance of samedetergent composition without the enzyme e.g. by increased stain removalor less re-deposition. The term “improved wash performance” includeswash performance in laundry.

Laundering: The term “laundering” relates to both household launderingand industrial laundering and means the process of treating textileswith a solution containing a cleaning or detergent composition of thepresent invention. The laundering process can for example be carried outusing e.g. a household or an industrial washing machine or can becarried out by hand.

Malodor: By the term “malodor” is meant an odor which is not desired onclean items. The cleaned item should smell fresh and clean withoutmalodors adhered to the item. One example of malodor is compounds withan unpleasant smell, which may be produced by microorganisms. Anotherexample is unpleasant smells can be sweat or body odor adhered to anitem which has been in contact with human or animal. Another example ofmalodor can be the odor from spices, which sticks to items for examplecurry or other exotic spices which smells strongly.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In some aspects, the maturepolypeptide is amino acids 1 to 324 of SEQ ID NO 2 and amino acids −41to −1 of SEQ ID NO 2 are a signal peptide. In some aspects, the maturepolypeptide is the amino acid sequence having SEQ ID NO 7. In someaspects, the mature polypeptide is amino acids 1 to 324 of SEQ ID NO 4and amino acids −25 to −1 of SEQ ID NO 4 are a signal peptide. In someaspects, the mature polypeptide is the amino acid sequence having SEQ IDNO 8. In some aspects, the mature polypeptide is amino acids 1 to 324 ofSEQ ID NO 6 and amino acids −25 to −1 of SEQ ID NO 6 are a signalpeptide. In some aspects, the mature polypeptide is the amino acidsequence having SEQ ID NO 9. In some aspects, the mature polypeptide isamino acids 1 to 324 of SEQ ID NO 13 and amino acids −24 to −1 of SEQ IDNO 13 are a signal peptide. In some aspects, the mature polypeptide isthe amino acid sequence having SEQ ID NO 10. In some aspects, the maturepolypeptide is amino acids 1 to 324 of SEQ ID NO 15 and amino acids −24to −1 of SEQ ID NO 15 are a signal peptide. In some aspects, the maturepolypeptide is the amino acid sequence having SEQ ID NO 11.

It is known in the art that a host cell may produce a mixture of two ofmore different mature polypeptides (i.e., with a different C-terminaland/or N-terminal amino acid) expressed by the same polynucleotide. Itis also known in the art that different host cells process polypeptidesdifferently, and thus, one host cell expressing a polynucleotide mayproduce a different mature polypeptide (e.g., having a differentC-terminal and/or N-terminal amino acid) as compared to another hostcell expressing the same polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving hexosaminidase activity. In one aspect, the mature polypeptidecoding sequence is nucleotides 124 to 1095 of SEQ ID NO 1 andnucleotides 1 to 123 of SEQ ID NO 1 encodes a signal peptide. In oneaspect, the mature polypeptide coding sequence is nucleotides 76 to 1047of SEQ ID NO 3 and nucleotides 1 to 75 of SEQ ID NO 3 encode a signalpeptide. In one aspect, the mature polypeptide coding sequence isnucleotides 76 to 1047 of SEQ ID NO 5 and nucleotides 1 to 75 of SEQ IDNO 5 encode a signal peptide. In one aspect, the mature polypeptidecoding sequence is nucleotides 73 to 1044 of SEQ ID NO 12 andnucleotides 1 to 72 of SEQ ID NO 12 encode a signal peptide. In oneaspect, the mature polypeptide coding sequence is nucleotides 73 to 1044of SEQ ID NO 14 and nucleotides 1 to 72 of SEQ ID NO 14 encode a signalpeptide.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the −nobrief option) is usedas the percent identity and is calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

Stringency conditions: The term “very low stringency conditions” meansfor probes of at least 100 nucleotides in length, pre-hybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 25% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at45° C.

The term “low stringency conditions” means for probes of at least 100nucleotides in length, pre-hybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 25% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 2×SSC, 0.2% SDS at 50° C.

The term “medium stringency conditions” means for probes of at least 100nucleotides in length, pre-hybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 35% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 2×SSC, 0.2% SDS at 55° C.

The term “medium-high stringency conditions” means for probes of atleast 100 nucleotides in length, pre-hybridization and hybridization at42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denaturedsalmon sperm DNA, and 35% formamide, following standard Southernblotting procedures for 12 to 24 hours. The carrier material is finallywashed three times each for 15 minutes using 2×SSC, 0.2% SDS at 60° C.

The term “high stringency conditions” means for probes of at least 100nucleotides in length, pre-hybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 50% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 2×SSC, 0.2% SDS at 65° C.

The term “very high stringency conditions” means for probes of at least100 nucleotides in length, pre-hybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 50% formamide, following standard Southern blottingprocedures for 12 to 24 hours. The carrier material is finally washedthree times each for 15 minutes using 2×SSC, 0.2% SDS at 70° C.]

Variant: The term “variant” means a polypeptide having hexosaminidaseactivity comprising an alteration, i.e., a substitution, insertion,and/or deletion, at one or more (e.g., several) positions. Asubstitution means replacement of the amino acid occupying a positionwith a different amino acid; a deletion means removal of the amino acidoccupying a position; and an insertion means adding an amino acidadjacent to and immediately following the amino acid occupying aposition.

Nomenclature

For purposes of the present invention, the nomenclature [IV] or [I/V]means that the amino acid at this position may be isoleucine (Ile, I) orvaline (Val, V). Likewise, the nomenclature [LVI] and [L/V/I] means thatthe amino acid at this position may be a leucine (Leu, L), valine (Val,V) or isoleucine (Ile, I), and so forth for other combinations asdescribed herein. Unless otherwise limited further, the amino acid X isdefined such that it may be any of the 20 natural amino acids.

DETAILED DESCRIPTION OF THE INVENTION

Various enzymes have been used in laundry many of which relate toremoval of malodor. WO2014/087011 describes the use of adeoxyribonuclease (DNase) for reducing malodor from laundry and/ortextile, WO9909143 describes the use of one or more oxidoreductases incombination with a mediator for the reduction of malodor andWO2012/112718 describe a method for inhibiting production of laundrymalodor caused by bacteria by using various strains of Bacillus. Thepresent invention relates to polypeptides and cleaning e.g. detergentcompositions comprising polypeptides from the Terribacillus clade ofGH20 family of polypeptides having hexosaminidase activity. Also claimedare laundering methods and the use of the polypeptides withhexosaminidase activity. The polypeptides from the Terribacillus cladeof GH20 family with hexosaminidase activity are useful in reducing andpreventing staining of items being washed. The inventors havesurprisingly found that the polypeptides of the Terribacillus clade ofGH20 family having hexosaminidase activity are useful for reduction oflaundry associated organic matter e.g. EPS and or PNAG. WO200406117describes compositions comprising dispersins e.g. DspB. The compositionmay include a detergent which may be anionic, cationic, or non-ionic.WO9850512 describes enzymes having hexosaminidase activity. Thepolypeptides of the present invention have hexosaminidase activityhowever, these do not belong to the DspB clade or to is related to anyof the enzymes described in WO9850512. The polypeptides of the inventionbelong to the Terribacillus clade and are thus distinct from the DspBand from those disclosed in WO9850512. It was therefore surprising thatthe polypeptide of the invention could be used for prevention, reductionand/or removal of organic matter such as biofilm components e.g.polysaccharides e.g. PNAG (poly-N-acetylglucosamine) on hard surfaces aswell as in laundry. There is no indication in the art of the use ofhexosaminidases in cleaning processes such as laundry or in detergentcompositions comprising e.g. builders and/or bleaches. To be useful incleaning processes the enzymes need to perform its action in detergentsunder the conditions of cleaning processes such as laundry, whichincludes stability in the presence of detergent components such assurfactants, builders and bleach components. The components of adetergent may significantly effect on the performance of the enzymessuch as the hexosaminidases. The present application surprisingly showsthat polypeptides belonging to the Terribacillus clade are particularlystable in the present of surfactant linear alkylbenzenesulfonates (LAS)which is a very common surfactant in detergents. The polypeptidesbelonging to the Terribacillus are thus particularly useful for deepcleaning e.g. of textiles or washing machines.

An overview of the Terribacillus clade is provided in FIG. 1. TheTerribacillus clade comprises homologous sequences. The polypeptideswith hexosaminidase activity of the present invention having the matureamino acid sequences SEQ ID 7, 8 and 9 can be pairwise aligned using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453). The percent identities resulting from such alignments areshown in Table 1 below.

TABLE 1 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO 11 NO 10 NO 7 NO 9 NO 8100 75.9 81.8 82.4 81.2 SEQ ID NO 11 75.9 100 77.5 76.5 77.5 SEQ ID NO10 81.8 77.5 100 95.1 95.7 SEQ ID NO 7 82.4 76.5 95.1 100 93.5 SEQ ID NO9 81.2 77.5 95.7 93.5 100 SEQ ID NO 8

Table 1 shows that the polypeptides of the invention share closesequence relatedness. The polypeptides comprising the amino acidssequences of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 and SEQID NO 11 belongs to a subclade of the Dispersin clade. Thesepolypeptides share more than 90% pairwise sequence identity and arecloser related to each other compared to e.g. the DspB polypeptideswhich lies further away (not shown).

One aspect of the invention relates to a polypeptide comprising theamino acid sequence shown in SEQ ID NO 7 or is a polypeptide having atleast 99.8% or 100% sequence identity hereto, wherein the polypeptidehas hexosaminidase activity.

One aspect of the invention relates to a polypeptide comprising theamino acid sequence shown in SEQ ID NO 8 or is a polypeptide having atleast 96%, such as at least 97%, such as at least 98%, such as at least99% or 100% sequence identity hereto, wherein the polypeptide hashexosaminidase activity.

One aspect of the invention relates to a polypeptide comprising theamino acid sequence shown in SEQ ID NO 9 or is a polypeptide having atleast 95%, such as at least 96%, such as at least 97%, such as at least98%, such as at least 99% or 100% sequence identity hereto, wherein thepolypeptide has hexosaminidase activity.

One aspect of the invention relates to a polypeptide comprising theamino acid sequence shown in SEQ ID NO 10 or is a polypeptide having atleast 96%, such as at least 97%, such as at least 98%, such as at least99% or 100% sequence identity hereto, wherein the polypeptide hashexosaminidase activity.

One aspect of the invention relates to a polypeptide comprising theamino acid sequence shown in SEQ ID NO 11 or is a polypeptide having atleast 84%, such as at least 85%, such as at least 86%, such as at least87%, such as at least 88%, such as at least 89%, such as at least 90%,such as at least 91%, such as at least 92%, such as at least 93%, suchas at least 94%, such as at least 95%, such as at least 96%, such as atleast 97%, such as at least 98%, such as at least 99% or 100% sequenceidentity hereto, wherein the polypeptide has hexosaminidase activity.

The polypeptides of the invention all lies within the same clade, theTerribacillus clade, and all have common functional features includingdeep cleaning properties in the presence of detergents. The inventorshave surprisingly found that the polypeptides of the Terribacillus cladecomprising the mature polypeptides with SEQ ID NO 7, 8, 9, 10, 11 orpolypeptides having at least 80% sequence identity hereto, sharespecific properties, more precisely the polypeptides comprised by thisclade all have good deep cleaning effects with a broad range ofsurfactants such as LAS and are therefore particularly useful indetergents with surfactants, such as in detergent comprising anionic,non-ionic, cationic and/or amphoteric surfactants.

As already described the polypeptides of the invention havinghexosaminidase activity may comprise the structural domains ofGlyco_hydro_20 e.g. GH20. Polypeptides comprising a GH20 domain maycomprise several motifs one example is GXDE (SEQ ID NO 18) situated inpositions corresponding to positions 158 to 161 in Terribacillussaccharophilus (SEQ ID NO 9). Residues D and E are the key catalyticresidues of GH20 (pos 160 and 161 in SEQ ID NO 9). The GH20 polypeptidescan be separated into multiple distinct sub-clusters, or clades,examples of specific domains are listed below. A further domain,preferably shared by the polypeptides of the invention, was identified.This domain has not been described previously, the domain is termed IASand polypeptides of this domain are in addition to having hexosaminidaseactivity e.g. PNAG activity, characterized by comprising certain motifse.g. one or more of the [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQID NO 19), corresponding to ESYAIAS at position 44 to 50 of SEQ ID NO 9.Another domain, preferably shared by the polypeptides of the invention,was identified. This domain has not been described previously. Thedomain is termed WND and polypeptides of this domain comprise GH20domain, are of bacterial origin and are in addition to having PNAGactivity, characterized by comprising certain motifs. The polypeptidesof the domain may comprise the motif [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ IDNO: 20), corresponding to pos 156 to 163 of SEQ ID NO 9, where G(corresponding to position 158 of SEQ ID NO 9) is fully conserved inTerribacillus clade and residues D and E are the key catalytic residuesof GH20 (pos 160 and 161 in SEQ ID NO 9). Another motif which may becomprised by the polypeptides of the invention is WND[SQR][IVL][TLVM](SEQ ID NO: 21), 193 to 198 in SEQ ID NO 9, where W (pos 193 in SEQ IDNO 9) is part of the active site pocket and putatively involved inbinding of the N-acetyl group of the PNAG substrate. The polypeptides ofthe Terribacillus clade may be further subdivided in a clade termedQSTL, which comprises WND domain polypeptides of bacterial origin,having PNAG activity. The polypeptides of the clade comprise the motifexample QSTL (SEQ ID NO: 22), corresponding to pos 216 to 219 of SEQ IDNO 9, where all four amino are fully conserved in QSTL clade andputatively involved in substrate binding. Another motif which may becomprised by the polypeptides of the QSTL clade is NKFFY (SEQ ID NO:23), 273 to 277 in SEQ ID NO 9. A further motif which may be comprisedby the polypeptides of the QSTL clade is NLD[DR]S (SEQ ID NO: 24), 204to 208 in SEQ ID NO 9. In one aspect, the polypeptides of the inventioncomprise the GXDE (SEQ ID NO 18) motif. In one aspect, the polypeptidesof the invention comprise the [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN](SEQ ID NO 19) motif. In one aspect, the polypeptides of the inventioncomprise the motif [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO: 20). In oneaspect, the polypeptides of the invention comprise the motifWND[SQR][IVL][TLVM] (SEQ ID NO: 21). In one aspect, the polypeptides ofthe invention comprise the motif QSTL (SEQ ID NO 22). In one aspect, thepolypeptides of the invention comprise the motif NKFFY (SEQ ID NO: 23).In one aspect, the polypeptides of the invention comprise the motifNLD[DR]S (SEQ ID NO: 24). An alignment of the polypeptides of theinvention comprised in the QSTL clade is shown in FIG. 2.

Some aspect of the invention relates to a polypeptide comprising one orboth motif(s) GXDE (SEQ ID NO 18) or[EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19), wherein thepolypeptide comprises an amino acid sequence selected from thepolypeptides shown in SEQ ID NOS 7, 8, 9, 10 and 11 or polypeptideshaving at least 80% sequence identity hereto.

Some aspect of the invention relates to a polypeptide comprising one orboth motif(s) [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO 20) orWND[SQR][IVL][TLVM] (SEQ ID NO 21), wherein the polypeptide comprises anamino acid sequence selected from the polypeptides shown in SEQ ID NOS7, 8, 9, 10 and 11 or polypeptides having at least 80% sequence identityhereto.

Some aspect of the invention relates to a polypeptide comprising one ormore of the motif(s) QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO: 23) orNLD[DR]S (SEQ ID NO: 24), wherein the polypeptide comprises an aminoacid sequence selected from the polypeptides shown in SEQ ID NOS 7, 8,9, 10 and 11 or polypeptides having at least 80% sequence identityhereto.

One aspect of the invention relates to a polypeptide comprising one orboth motif(s) [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO 20) orWND[SQR][IVL][TLVM] (SEQ ID NO 21), wherein the polypeptide comprisesthe amino acid sequence shown in SEQ ID NO 7 or is a polypeptide havingat least 84%, such as at least 85%, such as at least 86%, such as atleast 87%, such as at least 88%, such as at least 89%, such as at least90%, such as at least 91%, such as at least 92%, such as at least 93%,such as at least 94%, such as at least 95%, such as at least 96%, suchas at least 97%, such as at least 98%, such as at least 99% or 100%sequence identity hereto, wherein the polypeptide has hexosaminidaseactivity.

One aspect of the invention relates to a polypeptide comprising one orboth motif(s) [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO 20) orWND[SQR][IVL][TLVM] (SEQ ID NO 21), wherein the polypeptide comprisesthe amino acid sequence shown in SEQ ID NO 8 or is a polypeptide havingat least 84%, such as at least 85%, such as at least 86%, such as atleast 87%, such as at least 88%, such as at least 89%, such as at least90%, such as at least 91%, such as at least 92%, such as at least 93%,such as at least 94%, such as at least 95%, such as at least 96%, suchas at least 97%, such as at least 98%, such as at least 99% or 100%sequence identity hereto, wherein the polypeptide has hexosaminidaseactivity.

One aspect of the invention relates to a polypeptide comprising one orboth motif(s) [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO 20) orWND[SQR][IVL][TLVM] (SEQ ID NO 21), wherein the polypeptide comprisesthe amino acid sequence shown in SEQ ID NO 9 or is a polypeptide havingat least 84%, such as at least 85%, such as at least 86%, such as atleast 87%, such as at least 88%, such as at least 89%, such as at least90%, such as at least 91%, such as at least 92%, such as at least 93%,such as at least 94%, such as at least 95%, such as at least 96%, suchas at least 97%, such as at least 98%, such as at least 99% or 100%sequence identity hereto, wherein the polypeptide has hexosaminidaseactivity.

One aspect of the invention relates to a polypeptide comprising one orboth motif(s) [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO 20) orWND[SQR][IVL][TLVM] (SEQ ID NO 21), wherein the polypeptide comprisesthe amino acid sequence shown in SEQ ID NO 10 or is a polypeptide havingat least 84%, such as at least 85%, such as at least 86%, such as atleast 87%, such as at least 88%, such as at least 89%, such as at least90%, such as at least 91%, such as at least 92%, such as at least 93%,such as at least 94%, such as at least 95%, such as at least 96%, suchas at least 97%, such as at least 98%, such as at least 99% or 100%sequence identity hereto, wherein the polypeptide has hexosaminidaseactivity.

One aspect of the invention relates to a polypeptide comprising one orboth motif(s) [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO 20) orWND[SQR][IVL][TLVM] (SEQ ID NO 21), wherein the polypeptide comprisesthe amino acid sequence shown in SEQ ID NO 11 or is a polypeptide havingat least 84%, such as at least 85%, such as at least 86%, such as atleast 87%, such as at least 88%, such as at least 89%, such as at least90%, such as at least 91%, such as at least 92%, such as at least 93%,such as at least 94%, such as at least 95%, such as at least 96%, suchas at least 97%, such as at least 98%, such as at least 99% or 100%sequence identity hereto, wherein the polypeptide has hexosaminidaseactivity.

One aspect of the invention relates to a polypeptide comprising one ormore of the motif(s) QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO: 23) orNLD[DR]S (SEQ ID NO: 24), wherein the polypeptide comprises the aminoacid sequence shown in SEQ ID NO 7 or is a polypeptide having at least84%, such as at least 85%, such as at least 86%, such as at least 87%,such as at least 88%, such as at least 89%, such as at least 90%, suchas at least 91%, such as at least 92%, such as at least 93%, such as atleast 94%, such as at least 95%, such as at least 96%, such as at least97%, such as at least 98%, such as at least 99% or 100% sequenceidentity hereto, wherein the polypeptide has hexosaminidase activity.

One aspect of the invention relates to a polypeptide comprising one ormore of the motif(s) QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO: 23) orNLD[DR]S (SEQ ID NO: 24), wherein the polypeptide comprises the aminoacid sequence shown in SEQ ID NO 8 or is a polypeptide having at least84%, such as at least 85%, such as at least 86%, such as at least 87%,such as at least 88%, such as at least 89%, such as at least 90%, suchas at least 91%, such as at least 92%, such as at least 93%, such as atleast 94%, such as at least 95%, such as at least 96%, such as at least97%, such as at least 98%, such as at least 99% or 100% sequenceidentity hereto, wherein the polypeptide has hexosaminidase activity.

One aspect of the invention relates to a polypeptide comprising one ormore of the motif(s) QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO: 23) orNLD[DR]S (SEQ ID NO: 24), wherein the polypeptide comprises the aminoacid sequence shown in SEQ ID NO 9 or is a polypeptide having at least84%, such as at least 85%, such as at least 86%, such as at least 87%,such as at least 88%, such as at least 89%, such as at least 90%, suchas at least 91%, such as at least 92%, such as at least 93%, such as atleast 94%, such as at least 95%, such as at least 96%, such as at least97%, such as at least 98%, such as at least 99% or 100% sequenceidentity hereto, wherein the polypeptide has hexosaminidase activity.

One aspect of the invention relates to a polypeptide comprising one ormore of the motif(s) QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO: 23) orNLD[DR]S (SEQ ID NO: 24), wherein the polypeptide comprises the aminoacid sequence shown in SEQ ID NO 10 or is a polypeptide having at least84%, such as at least 85%, such as at least 86%, such as at least 87%,such as at least 88%, such as at least 89%, such as at least 90%, suchas at least 91%, such as at least 92%, such as at least 93%, such as atleast 94%, such as at least 95%, such as at least 96%, such as at least97%, such as at least 98%, such as at least 99% or 100% sequenceidentity hereto, wherein the polypeptide has hexosaminidase activity.

One aspect of the invention relates to a polypeptide comprising one ormore of the motif(s) QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO: 23) orNLD[DR]S (SEQ ID NO: 24), wherein the polypeptide comprises the aminoacid sequence shown in SEQ ID NO 11 or is a polypeptide having at least84%, such as at least 85%, such as at least 86%, such as at least 87%,such as at least 88%, such as at least 89%, such as at least 90%, suchas at least 91%, such as at least 92%, such as at least 93%, such as atleast 94%, such as at least 95%, such as at least 96%, such as at least97%, such as at least 98%, such as at least 99% or 100% sequenceidentity hereto, wherein the polypeptide has hexosaminidase activity.

One aspect, relates to the use in a cleaning process of a polypeptidecomprising the amino acid sequence shown in SEQ ID NO 7, SEQ ID NO 8,SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 or is a polypeptide having atleast 84%, such as at least 85%, such as at least 86%, such as at least87%, such as at least 88%, such as at least 89%, such as at least 90%,such as at least 91%, such as at least 92%, such as at least 93%, suchas at least 94%, such as at least 95%, such as at least 96%, such as atleast 97%, such as at least 98%, such as at least 99% or 100% sequenceidentity hereto, wherein the polypeptide has hexosaminidase activity.Some aspects of the invention relate to cleaning e.g detergentcompositions comprising a) one or more polypeptide selected from thegroup consisting of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10and SEQ ID NO 11, wherein the polypeptide has hexosaminidase activityand b) at least one surfactant, preferably at least one surfactantselected from the group consisting of anionic, nonionic and/or cationicsurfactants.

On aspect of the invention relates to a composition comprising at least0.001 ppm polypeptide having hexosaminidase activity, wherein thepolypeptide is selected from the group consisting of polypeptides havingat least 60% sequence identity to the mature polypeptide shown in SEQ IDNO 7, 8, 9, 10 and 11; and at least one adjunct ingredient.

The amount of polypeptide may be in the range of 0.00004-100 ppm, suchas in the range of 0.00008-50 ppm, in the range of 0.00001-20, in therange of 0.0002-20 ppm, in the range of 0.0001-50 ppm, in the range of0.0002-50, in the range of 0.0004-50, in the range of 0.0008-50, in therange of 0.001-50 ppm, 0.01-50 ppm, preferably 0.0001-50 ppm, morepreferably 0.0002-20 ppm, more preferably 0.0002-10 ppm, more preferably0.001-10 ppm, and most preferably 0.002-10 ppm. The hexosaminidase ofthe present invention may be in an amount corresponding to at least0.00001 ppm, such as at least 0.00002 ppm, at least 0.0001 ppm, at least0.0002 ppm, at least 0.0005 ppm, at least 0.001 ppm, at least 0.002 mgppm, at least 0.005 ppm, at least 0.01 ppm or at least 0.02 ppm. Thecomposition may comprise at least 0.00008%, preferably at least0.0000.1%, 0.00002%, 0.000.1%, 0.0002%, 0.001%, 0.002%, 0.003%, 0.004%,0.005%, 0.006%, 0.008%, 0.01%, 0.02%, 0.03%, 0.05%, 0.1%, 0.2%, 0.3%,0.4%, 0.6%, 0.7%, 0.8%, 0.9% or 1.0% hexosaminidase.

Preferably, the polypeptide has N-acetylglucosaminidase activity and/orβ-N-acetylglucosamininidase activity e.g. activity to PNAG. In oneaspect, the polypeptide comprises one or more of the motif(s) GXDE (SEQID NO 18), [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19),[VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO: 20), WND[SQR][IVL][TLVM] (SEQ IDNO: 21), QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO: 23), NLD[DR]S (SEQ IDNO: 24). In one aspect, the polypeptide has N-acetylglucosaminidaseactivity and/or β-N-acetylglucosamininidase activity e.g. activity toPNAG and comprises one or more of the motif(s) GXDE (SEQ ID NO 18),[EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19),[VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO: 20), WND[SQR][IVL][TLVM] (SEQ IDNO: 21), QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO: 23), NLD[DR]S (SEQ IDNO: 24). In one aspect, the polypeptide has at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or 100% sequenceidentity to the polypeptide shown in SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO9, SEQ ID NO 10 and SEQ ID NO 11.

In one aspect, the polypeptide has N-acetylglucosaminidase activityand/or β-N-acetylglucosamininidase activity e.g. activity to PNAG andcomprises one or more of the motif(s) GXDE (SEQ ID NO 18),[EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19),[VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO: 20), WND[SQR][IVL][TLVM] (SEQ IDNO: 21), QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO: 23), NLD[DR]S (SEQ IDNO: 24), wherein the polypeptide has at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or 100% sequenceidentity to the polypeptide shown in SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO9, SEQ ID NO 10 and SEQ ID NO 11.

In one aspect, the polypeptide comprising or consisting of SEQ ID NO 7or the mature polypeptide of SEQ ID NO 2, comprising or consisting ofSEQ ID NO 8 or the mature polypeptide of SEQ ID NO 4, comprising orconsisting of SEQ ID NO 9 or the mature polypeptide of SEQ ID NO 6,comprising or consisting of SEQ ID NO 10 or the mature polypeptide ofSEQ ID NO 13 or comprising or consisting of SEQ ID NO 11 or the maturepolypeptide of SEQ ID NO 15.

The composition is preferably a cleaning composition preferably alaundry or dish wash composition. Some aspect of the invention relatesto a composition comprising a polypeptide of the invention and anadjunct ingredient, wherein the adjunct ingredient is selected from,

a) at least one builder,

b) at least one surfactant, and

at least one bleach component.

Some aspects of the invention relate to cleaning compositions e.g.detergent compositions comprising a) one or more polypeptide selectedfrom the group consisting of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQID NO 10 and SEQ ID NO 11 or a polypeptide having at least 80% sequenceidentity hereto, wherein the polypeptide has hexosaminidase activity andb) at least one surfactant, preferably at least one surfactant selectedfrom the group consisting of anionic, nonionic and/or cationicsurfactants.

Some aspect of the invention relates to a detergent compositioncomprising:

-   -   a) at least 0.01 ppm of active enzyme polypeptide, wherein the        enzyme polypeptide is selected from the group consisting of SEQ        ID NO 7, SEQ ID NO 8 or SEQ ID NO 9 or polypeptides having at        least 80% sequence identity hereto, wherein the polypeptide has        hexosaminidase activity, and    -   b) from about 2 wt % to about 60 wt % surfactant or from about 5        wt % to about 60 wt % surfactant.        Some aspect of the invention relates to a detergent composition        comprising:    -   a) at least 0.0001 ppm of active enzyme polypeptide, wherein the        enzyme polypeptide is selected from the group consisting of SEQ        ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11        or polypeptides having at least 80% sequence identity hereto,        wherein the polypeptide has hexosaminidase activity, and    -   b) from about 2 wt % to about 60 wt % surfactant or from about 5        wt % to about 60 wt % surfactant from about 5 wt % to about 60        wt % surfactant.        The composition preferably comprises from about 2 wt % to about        60 wt %, from about 5 wt % to about 50 wt %, from about 5 wt %        to about 40 wt %, from about 5 wt % to about 30 wt %, from about        5 wt % to about 20 wt %, from about 5 wt % to about 10 wt %        anionic surfactants and/or non-ionic surfactants. The surfactant        may be selected among nonionic, anionic and/or amphoteric        surfactants as described above, preferably anionic or nonionic        surfactants but also amphoteric surfactants may be used. In        general, bleach-stable surfactants are preferred. Preferred        anionic surfactants are sulphate surfactants and in particular        alkyl ether sulphates, especially C9-C15 alcohol ethersulfates,        C12-C15 primary alcohol ethoxylate, C8-C16 ester sulphates and        C10-C14 ester sulphates, such as mono dodecyl ester sulphates        Non-limiting examples of anionic surfactants include sulfates        and sulfonates, in particular, linear alkylbenzenesulfonates        (LAS), isomers of LAS, branched alkylbenzenesulfonates (BABS),        phenylalkanesulfonates, alpha-olefinsulfonates (AOS), olefin        sulfonates, alkene sulfonates, alkane-2,3-diylbis(sulfates),        hydroxyalkanesulfonates and disulfonates, alkyl sulfates (AS)        such as sodium dodecyl sulfate (SDS), fatty alcohol sulfates        (FAS), primary alcohol sulfates (PAS), alcohol ethersulfates        (AES or AEOS or FES, also known as alcohol ethoxysulfates or        fatty alcohol ether sulfates), secondary alkanesulfonates (SAS),        paraffin sulfonates (PS), ester sulfonates, sulfonated fatty        acid glycerol esters, alpha-sulfo fatty acid methyl esters        (alpha-SFMe or SES) including methyl ester sulfonate (MES),        alkyl- or alkenylsuccinic acid, dodecenyl/tetradecenyl succinic        acid (DTSA), fatty acid derivatives of amino acids, diesters and        monoesters of sulfo-succinic acid or salt of fatty acids (soap),        and combinations thereof. The anionic surfactants are preferably        added to the detergent in the form of salts. Suitable cations in        these salts are alkali metal ions, such as sodium, potassium and        lithium and ammonium salts, for example (2-hydroxyethyl)        ammonium, bis(2-hydroxyethyl) ammonium and tris(2-hydroxyethyl)        ammonium salts. Non-limiting examples of nonionic surfactants        include alcohol ethoxylates (AE or AEO), alcohol propoxylates,        propoxylated fatty alcohols (PFA), alkoxylated fatty acid alkyl        esters, such as ethoxylated and/or propoxylated fatty acid alkyl        esters, alkylphenol ethoxylates (APE), nonylphenol ethoxylates        (NPE), alkylpolyglycosides (APG), alkoxylated amines, fatty acid        monoethanolamides (FAM), fatty acid diethanolamides (FADA),        ethoxylated fatty acid monoethanolamides (EFAM), propoxylated        fatty acid monoethanolamides (PFAM), polyhydroxyalkyl fatty acid        amides, or N-acyl N-alkyl derivatives of glucosamine        (glucamides, GA, or fatty acid glucamides, FAGA), as well as        products available under the trade names SPAN and TWEEN, and        combinations thereof. Commercially available nonionic        surfactants includes Plurafac™, Lutensol™ and Pluronic™ range        from BASF, Dehypon™ series from Cognis and Genapol™ series from        Clariant.        In some aspect of the invention a detergent composition of the        invention comprises:

a) at least 0.01 ppm of active enzyme polypeptide, wherein the enzymepolypeptide belongs to the Terribacillus clade and is selected from thegroup consisting of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10and SEQ ID NO 11 or polypeptides having at least 60% sequence identityhereto, wherein the polypeptide has hexosaminidase activity,

b) from about 2 wt % to about 60 wt % of at least one surfactant

In a preferred aspect of the invention the ratio of anionic/nonionicsurfactant is above 1 i.e. the content of anionic surfactant is higherthan the amount of nonionic surfactant. Thus, one aspect of theinvention relates to a detergent composition comprising:

-   -   a) at least 0.01 ppm of active enzyme polypeptide, wherein the        enzyme polypeptide belongs to the Terribacillus clade and is        selected from the group consisting of SEQ ID NO 7, SEQ ID NO 8,        SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11 or polypeptides        having at least 60% sequence identity hereto, wherein the        polypeptide has hexosaminidase activity,    -   b) from about 5 wt % to about 50 wt % anionic surfactants, and    -   c) from about 1 wt % to about 8 wt % nonionic surfactants.        The polypeptides of the invention may also be formulated in        compositions e.g. liquid laundry composition optionally        comprising a builder e.g. a liquid laundry compositions        comprising:    -   a) at least 0.0001 ppm e.g. 0.01 ppm of active enzyme        polypeptide, wherein the enzyme polypeptide belongs to the        Terribacillus clade and is selected from the group consisting of        SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 and SEQ ID        NO 11 or polypeptides having at least 60% sequence identity        hereto, wherein the polypeptide has hexosaminidase activity,    -   b) from about 2 wt % to about 60 wt % of at least one        surfactant, and optionally    -   c) from about 5 wt % to about 50 wt % of at least one builder        such as carbonates, zeolites, phosphate builder, calcium        sequestering builders or complexing agents.

The composition comprises at least one builder, wherein the builder isadded in an amount from about 0 to about 65% wt %, from about 40 wt % toabout 65 wt %, from about 20 wt % to about 65 wt %, from about 10 wt %to about 50 wt % or from about 5 wt % to about 50 wt % weight, whereinthe builder is selected among phosphates, sodium citrate builders,sodium carbonate, sodium silicate, sodium and zeolites

One aspect of the invention relates to a cleaning compositioncomprising:

a) at least 0.0001 ppm polypeptide, wherein the polypeptide belongs tothe Terribacillus clade, comprises one or both motif(s)[VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO 20) or WND[SQR][IVL][TLVM] (SEQID NO 21), wherein the polypeptide is selected from the group consistingof SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 andpolypeptides having at least 80% sequence identity hereto and whereinthe polypeptide has hexosaminidase activity, and optionally

b) from about 2 wt % to about 60 wt % of at least one surfactant, andoptionally

c) from about 5 wt % to about 50 wt % of at least one builder, andoptionally

d) from about 1 wt % to about 15 wt % of at least on bleach component.

The surfactant may be any of those described above.

The builder is preferably selected among phosphates, sodium citratebuilders, sodium carbonate, sodium silicate, sodium aluminosilicate(zeolite). Suitable builders are alkali metal or ammonium phosphates,polyphosphates, phosphonates, polyphosphonates, carbonates,bicarbonates, borates, citrates, and polycarboxylates. Citrate builders,e.g., citric acid and soluble salts thereof (particularly sodium salt),are polycarboxylate builders. Citrates can be used in combination withzeolite, silicates like the BRITESIL types, and/or layered silicatebuilders. The builder is preferably added in an amount of about 0-65% byweight, such as about 5% to about 50% by weight. In the composition, thelevel of builder is typically about 40-65% by weight, particularly about50-65% by weight, particularly from 20% to 50% by weight. The builderand/or co-builder may particularly be a chelating agent that formswater-soluble complexes with Ca and Mg. Any builder and/or co-builderknown in the art for use in cleaning detergents may be utilized.Non-limiting examples of builders include zeolites, diphosphates(pyrophosphates), triphosphates such as sodium triphosphate (STP orSTPP), carbonates such as sodium carbonate, soluble silicates such assodium metasilicate, layered silicates (e.g., SKS-6 from Hoechst), and(carboxymethyl)inulin (CMI), and combinations thereof. Furthernon-limiting examples of builders include citrate, chelators such asaminocarboxylates, aminopolycarboxylates and phosphonates, and alkyl- oralkenylsuccinic acid. Additional specific examples include2,2′,2″-nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid(EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid(IDS), ethylenediamine-N,N′-disuccinic acid (EDDS),methylglycine-N,N-diacetic acid (MGDA), glutamic acid-N,N-diacetic acid(GLDA), 1-hydroxyethane-1,1-diphosphonic acid,N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic acid-N-monoaceticacid (ASMA), aspartic acid-N,N-diacetic acid (ASDA), asparticacid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA),N-(sulfomethyl)aspartic acid (SMAS), N-(2-sulfoethyl)-aspartic acid(SEAS), N-(sulfomethylglutamic acid (SMGL), N-(2-sulfoethyl)-glutamicacid (SEGL), N-methyliminodiacetic acid (MIDA), serine-N,N-diacetic acid(SEDA), isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diaceticacid (PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilicacid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA) andN″-(2-hydroxyethyl)ethylenediamine-N,N,N′-triacetic acid (HEDTA),diethanolglycine (DEG), and combinations and salts thereof.

Phosphonates suitable for use herein include1-hydroxyethane-1,1-diphosphonic acid (HEDP), ethylenediaminetetrakis(methylenephosphonic acid) (EDTMPA), diethylenetriaminepentakis(methylenephosphonic acid) (DTMPA or DTPMPA or DTPMP), nitrilotris(methylenephosphonic acid) (ATMP or NTMP),2-phosphonobutane-1,2,4-tricarboxylic acid (PBTC),hexamethylenediaminetetrakis (methylenephosphonic acid) (HDTMP) Thecomposition may also contain 0-50% by weight, such as about 5% to about30%, of a detergent co-builder.

The composition may include a co-builder alone, or in combination with abuilder, for example a zeolite builder. Non-limiting examples ofco-builders include homopolymers of polyacrylates or copolymers thereof,such as poly (acrylic acid) (PAA) or copoly (acrylic acid/maleic acid)(PAA/PMA) or polyaspartic acid.

Further exemplary builders and/or co-builders are described in, e.g., WO09/102854, U.S. Pat. No. 5,977,053

In one preferred embodiment, the builder is a non-phosphorus basedbuilder such as citric acid and/or methylglycine-N, N-diacetic acid(MGDA) and/or glutamic-N, N-diacetic acid (GLDA) and/or salts thereof.

Some aspect of the invention relates a composition comprising at leastone enzyme polypeptide, wherein the enzyme is selected from the groupconsisting of polypeptides having SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9,SEQ ID NO 10 and SEQ ID NO 11 or polypeptides having at least 60%sequence identity hereto, wherein the polypeptide has hexosaminidaseactivity and a non-phosphate builder selected from citric acid, methylglycine-N,N-diacetic acid (MGDA) and/or glutamic-N,N-diacetic acid(GLDA) and mixtures thereof.

In one aspect, the composition is detergent composition, such as alaundry composition, an automatic dish wash composition (ADW)comprising:

a) at least 0.0001 ppm e.g. 0.01 ppm of active enzyme polypeptide,wherein the enzyme polypeptide is selected from the group consisting ofSEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11 orpolypeptides having at least 60% sequence identity hereto, wherein thepolypeptide has hexosaminidase activity, and

b) 10-50 wt % builder selected from citric acid, methylglycine-N,N-diacetic acid (MGDA) and/or glutamic acid-N, N-diacetic acid (GLDA)and mixtures thereof, and optionally

c) at least one bleach component.

The composition may contain 0-30% by weight, such as about 1% to about20%, such as about 1% to about 10%, such as about 1% to about 5%, suchas about 10% to about 30%, such as about 5% to about 10% or such asabout 10% to about 20% by weight (wt %) of a bleaching system. Anybleaching system comprising components known in the art for use incleaning detergents may be utilized. Suitable bleaching systemcomponents include sources of hydrogen peroxide; sources of peracids;and bleach catalysts or boosters.

Sources of Hydrogen Peroxide

Suitable sources of hydrogen peroxide are inorganic persalts, includingalkali metal salts such as sodium percarbonate and sodium perborates(usually mono- or tetrahydrate), and hydrogen peroxide-urea (1/1).

Sources of Peracids

Peracids may be (a) incorporated directly as preformed peracids or (b)formed in situ in the wash liquor from hydrogen peroxide and a bleachactivator (perhydrolysis) or (c) formed in situ in the wash liquor fromhydrogen peroxide and a perhydrolase and a suitable substrate for thelatter, e.g., an ester.

a) Suitable preformed peracids include, but are not limited to,peroxycarboxylic acids such as peroxybenzoic acid and itsring-substituted derivatives, peroxy-α-naphthoic acid, peroxyphthalicacid, peroxylauric acid, peroxystearic acid, ε-phthalimidoperoxycaproicacid [phthalimidoperoxyhexanoic acid (PAP)], ando-carboxybenzamidoperoxycaproic acid; aliphatic and aromaticdiperoxydicarboxylic acids such as diperoxydodecanedioic acid,diperoxyazelaic acid, diperoxysebacic acid, diperoxybrassylic acid,2-decyldiperoxybutanedioic acid, and diperoxyphthalic, -isophthalic and-terephthalic acids; perimidic acids; peroxymonosulfuric acid;peroxydisulfuric acid; peroxyphosphoric acid; peroxysilicic acid; andmixtures of said compounds. It is understood that the peracids mentionedmay in some cases be best added as suitable salts, such as alkali metalsalts (e.g., Oxone®) or alkaline earth-metal salts.

b) Suitable bleach activators include those belonging to the class ofesters, amides, imides, nitriles or anhydrides and, where applicable,salts thereof. Suitable examples are tetraacetylethylenediamine (TAED),sodium 4-[(3,5,5-trimethylhexanoyl) oxy] benzene-1-sulfonate (ISONOBS),sodium 4-(dodecanoyloxy) benzene-1-sulfonate (LOBS), sodium4-(decanoyloxy) benzene-1-sulfonate, 4-(decanoyloxy) benzoic acid(DOBA), sodium 4-(nonanoyloxy) benzene-1-sulfonate (NOBS), and/or thosedisclosed in WO98/17767. A family of bleach activators of interest wasdisclosed in EP624154 and particularly preferred in that family isacetyl triethyl citrate (ATC). ATC or a short chain triglyceride liketriacetin has the advantage that they are environmentally friendly.Furthermore, acetyl triethyl citrate and triacetin have goodhydrolytical stability in the product upon storage and are efficientbleach activators. Finally, ATC is multifunctional, as the citratereleased in the perhydrolysis reaction may function as a builder.

Bleach Catalysts and Boosters

The bleaching system may also include a bleach catalyst or booster.

Some non-limiting examples of bleach catalysts that may be used in thecompositions of the present invention include manganese oxalate,manganese acetate, manganese-collagen, cobalt-amine catalysts andmanganese triazacyclononane (MnTACN) catalysts; particularly preferredare complexes of manganese with 1,4,7-trimethyl-1,4,7-triazacyclononane(Me3-TACN) or 1,2,4,7-tetramethyl-1,4,7-triazacyclononane (Me4-TACN), inparticular Me3-TACN, such as the dinuclear manganese complex[(Me3-TACN)Mn(O)3Mn(Me3-TACN)](PF6)2, and[2,2′,2″-nitrilotris(ethane-1,2-diylazanylylidene-κN-methanylylidene)triphenolato-κ3O]manganese(III).The bleach catalysts may also be other metal compounds, such as iron orcobalt complexes.

In some embodiments, where a source of a peracid is included, an organicbleach catalyst or bleach booster may be used having one of thefollowing formulae:

(iii) and mixtures thereof; wherein each R1 is independently a branchedalkyl group containing from 9 to 24 carbons or linear alkyl groupcontaining from 11 to 24 carbons, preferably each R1 is independently abranched alkyl group containing from 9 to 18 carbons or linear alkylgroup containing from 11 to 18 carbons, more preferably each R1 isindependently selected from the group consisting of 2-propylheptyl,2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, dodecyl, tetradecyl,hexadecyl, octadecyl, isononyl, isodecyl, isotridecyl and isopentadecyl.Other exemplary bleaching systems are described, e.g. in WO2007/087258,WO2007/087244, WO2007/087259, EP1867708 (Vitamin K) and WO2007/087242.Suitable photobleaches may for example be sulfonated zinc or aluminiumphthalocyanines.

The present invention relates to polypeptides of the Terribacillus cladehaving hexosaminidase activity, compositions e.g. detergent compositionscomprising the polypeptides, and the use of detergent compositioncomprising the polypeptides of the invention for deep cleaning of anitem such as a textile.

Accordingly, some aspects of the invention relate to cleaningcomposition e.g. detergent compositions comprising:

-   -   a) at least 0.0001 ppm e.g. 0.001 ppm polypeptide of the        Terribacillus clade, optionally comprising one or more motif        selected from the group consisting of GXDE (SEQ ID NO 18),        [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19),        [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO: 20), WND[SQR][IVL][TLVM]        (SEQ ID NO: 21), QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO: 23),        NLD[DR]S (SEQ ID NO: 24), wherein the enzyme polypeptide is        selected from the group consisting of SEQ ID NO 7, SEQ ID NO 8,        SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 and polypeptides having        at least 60%, such as at least 70%, such as at least 80% or such        as at least 90% sequence identity hereto, wherein the        polypeptide has hexosaminidase activity, and optionally b) from        about 10 wt % to about 50 wt % builder preferably selected from        citric acid, methylglycine-N, N-diacetic acid (MGDA) and/or        glutamic acid-N, N-diacetic acid (GLDA) and mixtures thereof,        and optionally    -   c) from about 5 wt % to about 50 wt % surfactant, preferably        selected from anionic surfactants such as LAS, AOS, AEOS and/or        nonionic surfactants such as AE or AEO, and optionally    -   d) at least one bleach component, preferably selected from        percarbonates, persulphates and peracids.        Accordingly, some aspects of the invention relate to detergent        compositions comprising:    -   a) at least 0.0001 ppm e.g. 0.001 ppm polypeptide of the        Terribacillus clade, optionally comprising one or more motif        selected from the group consisting of GXDE (SEQ ID NO 18),        [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19),        [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO: 20), WND[SQR][IVL][TLVM]        (SEQ ID NO: 21), QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO: 23),        NLD[DR]S (SEQ ID NO: 24), wherein the enzyme polypeptide is        selected from the group consisting of SEQ ID NO 7, SEQ ID NO 8,        SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 and polypeptides having        at least 60%, such as at least 70%, such as at least 80% or such        as at least 90% sequence identity hereto, wherein the        polypeptide has hexosaminidase activity, and optionally    -   b) from about 10 wt % to about 50 wt % builder preferably        selected from citric acid, methylglycine-N, N-diacetic acid        (MGDA) and/or glutamic acid-N, N-diacetic acid (GLDA) and        mixtures thereof, and optionally    -   c) from about 5 wt % to about 50 wt % surfactant, preferably        selected from anionic surfactants such as LAS, AOS, AEOS and/or        nonionic surfactants such as AE or AEO, and optionally    -   d) at least one bleach component, preferably selected from        percarbonates, persulphates and peracids.        Accordingly, some aspects of the invention relate to detergent        compositions comprising:    -   a) at least 0.0001 ppm e.g. 0.001 ppm of polypeptide of the        Terribacillus clade, optionally comprising one or more motif        selected from the group consisting of GXDE (SEQ ID NO 18),        [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19),        [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO: 20), WND[SQR][IVL][TLVM]        (SEQ ID NO: 21), QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO: 23),        NLD[DR]S (SEQ ID NO: 24), wherein the enzyme polypeptide is        selected from the group consisting of SEQ ID NO 7, SEQ ID NO 8,        SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 and polypeptides having        at least 60%, such as at least 70%, such as at least 80% or such        as at least 90% sequence identity hereto, wherein the        polypeptide has hexosaminidase activity, and optionally    -   b) from about 10 wt % to about 50 wt % builder preferably        selected from citric acid, methylglycine-N, N-diacetic acid        (MGDA) and/or glutamic acid-N, N-diacetic acid (GLDA) and        mixtures thereof, and optionally    -   c) from about 5 wt % to about 50 wt % surfactant, preferably        selected from anionic surfactants such as LAS, AOS, AEOS and/or        nonionic surfactants such as AE or AEO, and optionally    -   d) at least one bleach component, wherein the bleach is a        peroxide and the bleach catalyst is a manganese compound,        wherein, the oxygen bleach is preferably percarbonate and the        manganese catalyst preferably        1,4,7-trimethyl-1,4,7-triazacyclononane or manganese (III)        acetate tetrahydrate (MnTACN).        One aspect relates to the use of a composition of as described        above for deep-cleaning of an item, such as a textile.

The polypeptides of the invention having hexosaminidase activity may beused for deep cleaning of items such as hard surfaces, textiles and/orfabric. In some aspects of the invention the polypeptides of theinvention e.g. the polypeptides having at least at least 60%, such as atleast 70%, such as at least 80% or such as at least 90% sequenceidentity the mature polypeptides of SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO6, SEQ ID NO 13 and SEQ ID NO 15 or to the mature polypeptide with SEQID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11, haveβ-N-acetylglucosamininidase activity and in some aspects thehexosaminidase activity is β-N-acetylglucosamininidase activity and thepolypeptide of the invention are β-N-acetylglucosamininidases.

One aspect of the invention relates to the use of a polypeptide of theTerribacillus clade, wherein the polypeptide has hexosaminidaseactivity, preferably N-acetylglucosaminidase activity and/orβ-N-acetylglucosamininidase activity in a cleaning process, such aslaundry and/or dish wash.

One aspect of the invention relates to the use of a polypeptidecomprising one or more of the motif(s) GXDE (SEQ ID NO 18),[EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19),[VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO: 20), WND[SQR][IVL][TLVM] (SEQ IDNO: 21), QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO: 23), NLD[DR]S (SEQ IDNO: 24).

One aspect of the invention relates to the use of a polypeptide of theTerribacillus clade, wherein the polypeptide has hexosaminidaseactivity, preferably N-acetylglucosaminidase activity and/orβ-N-acetylglucosamininidase activity in a cleaning process, such aslaundry and/or dish wash wherein the polypeptide comprises one or moreof the motif(s) GXDE (SEQ ID NO 18),[EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19),[VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO: 20), WND[SQR][IVL][TLVM] (SEQ IDNO: 21), QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO: 23), NLD[DR]S (SEQ IDNO: 24).

One aspect of the present invention relates to the use of a polypeptideof the Terribacillus clade e.g. comprising one or both of the motifsGXDE (SEQ ID NO 18), [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ IDNO 19), [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO: 20), WND[SQR][IVL][TLVM](SEQ ID NO: 21), QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO: 23), NLD[DR]S(SEQ ID NO: 24), wherein the polypeptide has hexosaminidase activity andwherein the polypeptide is a polypeptide having at least 60% e.g. 80%,85%, 90% or 95% sequence identity to the mature polypeptides shown inSEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 or SEQ ID NO 11 fordeep cleaning of an item, wherein the item is a textile. In one aspectof the invention the polypeptide of the Terribacillus clade havinghexosaminidase activity is used for preventing, reducing or removing thestickiness of an item. In one aspect of the invention the polypeptide ofthe Terribacillus clade having hexosaminidase activity can further beused for pre-treating stains on textile such as textile.

One aspect of the invention relates to the use of a polypeptide of theTerribacillus clade having hexosaminidase activity for preventing,reducing or removing re-deposition of soil during a wash cycle.

Further, one aspect of the invention relates to the use of a polypeptideof the Terribacillus clade having hexosaminidase activity forpreventing, reducing or removing the adherence of soil to an item. Inone embodiment, the item is textile. When the soil does not adhere tothe item, the item appears cleaner. Thus, the invention further concernsthe use of a polypeptide of the Terribacillus clade havinghexosaminidase activity for maintaining or improving the whiteness ofthe item.

When items like T-shirts or sportswear are used, they are exposed tobacteria from the body of the user and from the rest of the environmentin which they are used. This may cause malodor on the item even afterthe item is washed. The present invention therefore also concernsremoval or reduction of malodor on textile. The malodor may be caused bybacteria producing compounds with an unpleasant smell. One example ofsuch unpleasant smelling compounds is E-2-nonenal. The malodor can bepresent on newly washed textile which is still wet. Or the malodor canbe present on newly washed textile, which has subsequently been dried.The malodor may also be present on textile, which has been stored forsome time after wash. The present invention relates to reduction orremoval of malodor such as E-2-nonenal from wet or dry textile.

One aspect of the invention relates to the use of a polypeptide of theTerribacillus clade e.g. comprising one or more motif(s) GXDE (SEQ ID NO18), [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19),[VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO: 20), WND[SQR][IVL][TLVM] (SEQ IDNO: 21), QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO: 23), NLD[DR]S (SEQ IDNO: 24), wherein the polypeptide has hexosaminidase activity, preferablyN-acetylglucosaminidase activity and/or β-N-acetylglucosamininidaseactivity and wherein the polypeptide is a polypeptide having at 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to the mature polypeptides shown in SEQ ID NO 7,SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 or SEQ ID NO 11,

-   -   (i) for preventing, reducing or removing stickiness of the item;    -   (ii) for pretreating stains on the item;    -   (iii) for preventing, reducing or removing redeposition of soil        during a wash cycle;    -   (iv) for preventing, reducing or removing adherence of soil to        the item;    -   (v) for maintaining or improving whiteness of the item;    -   (vi) for preventing, reducing or removal malodor from the item,        wherein the item is a textile.

A cleaning e.g. detergent composition according to the invention maycomprise at least one adjunct ingredient e.g. a detergent adjunct, thedetergent adjunct ingredient may be surfactants and builders and/orchelators such as those described above. The adjunct ingredients mayalso be any of the following flocculating aid, dye transfer inhibitors,enzymes, enzyme stabilizers, enzyme inhibitors, catalytic materials,bleach activators, hydrogen peroxide, sources of hydrogen peroxide,preformed peracids, polymeric dispersing agents, clay soilremoval/anti-redeposition agents, brighteners, suds suppressors, dyes,perfumes, structure elasticizing agents, fabric softeners, carriers,hydrotropes, builders and co-builders, fabric hueing agents,anti-foaming agents, dispersants, processing aids, and/or pigments.

In one embodiment, the detergent adjunct ingredient is a builder or aclay soil removal/anti-redeposition agent.

In one embodiment, detergent adjunct ingredient is an enzyme. The one ormore enzymes may be selected from the group consisting of proteases,lipases, cutinases, amylases, carbohydrases, cellulases, pectinases,mannanases, arabinases, galactanases, xylanases and oxidases.

In addition to the polypeptides having hexosaminidase activitycomprising the polypeptides of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9,SEQ ID NO 10, SEQ ID NO 11 or a polypeptide having hexosaminidaseactivity and having at least 60% sequence identity hereto the cleaningcomposition of the invention may further comprise cellulases. Suitablecellulases include those of bacterial or fungal origin. Chemicallymodified or protein engineered mutants are included. Suitable cellulasesinclude cellulases from the genera Bacillus, Pseudomonas, Humicola,Fusarium, Thielavia, Acremonium, e.g., the fungal cellulases producedfrom Humicola insolens, Myceliophthora thermophila and Fusariumoxysporum disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263, 5,691,178,5,776,757 and WO 89/09259. Especially suitable cellulases are thealkaline or neutral cellulases having color care benefits. Examples ofsuch cellulases are cellulases described in EP 0 495 257, EP 0 531 372,WO 96/11262, WO 96/29397, WO 98/08940. Other examples are cellulasepolypeptides such as those described in WO 94/07998, EP 0 531 315, U.S.Pat. Nos. 5,457,046, 5,686,593, 5,763,254, WO 95/24471, WO 98/12307 andPCT/DK98/00299. Example of cellulases exhibiting endo-beta-1,4-glucanaseactivity (EC 3.2.1.4) are those having described in WO02/099091. Otherexamples of cellulases include the family 45 cellulases described inWO96/29397, and especially polypeptides thereof having substitution,insertion and/or deletion at one or more of the positions correspondingto the following positions in SEQ ID NO 8 of WO 02/099091: 2, 4, 7, 8,10, 13, 15, 19, 20, 21, 25, 26, 29, 32, 33, 34, 35, 37, 40, 42, 42a, 43,44, 48, 53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 70, 72, 76, 79, 80, 82,84, 86, 88, 90, 91, 93, 95, 95d, 95h, 95j, 97, 100, 101, 102, 103, 113,114, 117, 119, 121, 133, 136, 137, 138, 139, 140a, 141, 143a, 145, 146,147, 150e, 150j, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160c,160e, 160k, 161, 162, 164, 165, 168, 170, 171, 172, 173, 175, 176, 178,181, 183, 184, 185, 186, 188, 191, 192, 195, 196, 200, and/or 20,preferably selected among P19A, G20K, Q44K, N48E, Q119H or Q146R.Commercially available cellulases include Celluzyme™ Celluclean andCarezyme™ (Novozymes NS), Clazinase™, and Puradax HA™ (GenencorInternational Inc.), and KAC-500(B)™ (Kao Corporation).

In addition to the polypeptides having hexosaminidase activitycomprising SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 SEQ ID NO 10, SEQ ID NO11 or a polypeptide having hexosaminidase activity and having at least60% sequence identity hereto the cleaning composition of the inventionmay further comprise proteases. Suitable proteases include those ofbacterial, fungal, plant, viral or animal origin e.g. vegetable ormicrobial origin. Microbial origin is preferred. Chemically modified orprotein engineered mutants are included. It may be an alkaline protease,such as a serine protease or a metalloprotease. A serine protease mayfor example be of the 51 family, such as trypsin, or the S8 family suchas subtilisin. A metalloproteases protease may for example be athermolysin from e.g. family M4 or other metalloprotease such as thosefrom M5, M7 or M8 families. The term “subtilases” refers to a sub-groupof serine protease according to Siezen et al., Protein Engng. 4 (1991)719-737 and Siezen et al. Protein Science 6 (1997) 501-523. Serineproteases are a subgroup of proteases characterized by having a serinein the active site, which forms a covalent adduct with the substrate.The subtilases may be divided into 6 sub-divisions, i.e. the Subtilisinfamily, the Thermitase family, the Proteinase K family, the Lantibioticpeptidase family, the Kexin family and the Pyrolysin family. Examples ofsubtilases are those derived from Bacillus such as Bacillus lentus, B.alkalophilus, B. subtilis, B. amyloliquefaciens, Bacillus pumilus andBacillus gibsonii described in; U.S. Pat. No. 7,262,042 and WO09/021867,and subtilisin lentus, subtilisin Novo, subtilisin Carlsberg, Bacilluslicheniformis, subtilisin BPN′, subtilisin 309, subtilisin 147 andsubtilisin 168 described in WO89/06279 and protease PD138 described in(WO93/18140). Other useful proteases may be those described inWO92/175177, WO01/016285, WO02/026024 and WO02/016547. Examples oftrypsin-like proteases are trypsin (e.g. of porcine or bovine origin)and the Fusarium protease described in WO89/06270, WO94/25583 andWO05/040372, and the chymotrypsin proteases derived from Cellumonasdescribed in WO05/052161 and WO05/052146. A further preferred proteaseis the alkaline protease from Bacillus lentus DSM 5483, as described forexample in WO95/23221, and variants thereof which are described inWO92/21760, WO95/23221, EP1921147 and EP1921148. Examples ofmetalloproteases are the neutral metalloprotease as described inWO07/044993 (Genencor Int.) such as those derived from Bacillusamyloliquefaciens.

Examples of useful proteases are the variants described in: WO92/19729,WO96/034946, WO98/20115, WO98/20116, WO99/011768, WO01/44452,WO03/006602, WO04/03186, WO04/041979, WO07/006305, WO11/036263,WO11/036264, especially the variants with substitutions in one or moreof the following positions: 3, 4, 9, 15, 24, 27, 42, 55, 59, 60, 66, 74,85, 96, 97, 98, 99, 100, 101, 102, 104, 116, 118, 121, 126, 127, 128,154, 156, 157, 158, 161, 164, 176, 179, 182, 185, 188, 189, 193, 198,199, 200, 203, 206, 211, 212, 216, 218, 226, 229, 230, 239, 246, 255,256, 268 and 269 compared to SEQ ID NO 1 of WO2016/001449, wherein thepositions correspond to the positions of the Bacillus lentus proteaseshown in SEQ ID NO 1 of WO2016/001449. More preferred subtilase variantsmay comprise any of the the mutations: S3T, V41, S9R, S9E, A15T, S24G,S24R, K27R, N42R, S55P, G59E, G59D, N60D, N60E, V66A, N74D, N85S, N85R,G96S, G96A, S97G, S97D, S97A, S97SD, S99E, S99D, S99G, S99M, S99N, S99R,S99H, S101A, V1021, V102Y, V102N, S104A, G116V, G116R, H118D, H118N,N120S, S126L, P127Q, S128A, S154D, A156E, G157D, G157P, S158E, Y161A,R164S, Q176E, N179E, S182E, Q185N, A188P, G189E, V193M, N198D, V199I,Y203W, S206G, L211Q, L211D, N212D, N212S, M216S, A226V, K229L, Q230H,Q239R, N246K, N255W, N255D, N255E, L256E, L256D T268A or R269H. Theprotease variants are preferably variants of the Bacillus lentusprotease (Savinase®) shown in SEQ ID NO 1 of WO2016/001449 or theBacillus amylolichenifaciens protease (BPN′) shown in SEQ ID NO 2 ofWO2016/001449. The protease variants preferably have at least 80%sequence identity to SEQ ID NO 1 or SEQ ID NO 2 of WO2016/001449.

A protease variant comprising a substitution at one or more positionscorresponding to positions 171, 173, 175, 179, or 180 of SEQ ID NO 1 ofWO2004/067737, wherein said protease variant has a sequence identity ofat least 75% but less than 100% to SEQ ID NO 1 of WO2004/067737.

Suitable commercially available protease enzymes include those soldunder the trade names Alcalase®, Duralase™, Durazym™, Relase®, Relase®Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®,Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra,Blaze®, Blaze Evity® 100T, Blaze Evity® 125T, Blaze Evity® 150T,Neutrase®, Everlase® and Esperase® (Novozymes NS), those sold under thetradename Maxatase®, Maxacal®, Maxapem®, Purafect Ox®, Purafect OxP®,Puramax®, FN2®, FN3®, FN4®, Excellase®, Excellenz P1000™, ExcellenzP1250™, Eraser®, Preferenz P100™, Purafect Prime®, Preferenz P110™,Effectenz P1000™, Purafect®™, Effectenz P1050™, Purafect Ox®™, EffectenzP2000™, Purafast®, Properase®, Opticlean® and Optimase®(Danisco/DuPont), Axapem™ (Gist-Brocases N.V.), BLAP (sequence shown inFIG. 29 of U.S. Pat. No. 5,352,604) and variants hereof (Henkel AG) andKAP (Bacillus alkalophilus subtilisin) from Kao.

In addition to the polypeptides having hexosaminidase activitycomprising the polypeptides of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9,SEQ ID NO 10, SEQ ID NO 11 or a polypeptide having hexosaminidaseactivity and having at least 60% sequence identity hereto the cleaningcomposition of the invention may further comprise lipases and cutinaseswhich include those of bacterial or fungal origin. Chemically modifiedor protein engineered mutant enzymes are included. Examples includelipase from Thermomyces, e.g. from T. lanuginosus (previously namedHumicola lanuginosa) as described in EP258068 and EP305216, cutinasefrom Humicola, e.g. H. insolens (WO96/13580), lipase from strains ofPseudomonas (some of these now renamed to Burkholderia), e.g. P.alcaligenes or P. pseudoalcaligenes (EP218272), P. cepacia (EP331376),P. sp. strain SD705 (WO95/06720 & WO96/27002), P. wisconsinensis(WO96/12012), GDSL-type Streptomyces lipases (WO10/065455), cutinasefrom Magnaporthe grisea (WO10/107560), cutinase from Pseudomonasmendocina (U.S. Pat. No. 5,389,536), lipase from Thermobifida fusca(WO11/084412), Geobacillus stearothermophilus lipase (WO11/084417),lipase from Bacillus subtilis (WO11/084599), and lipase fromStreptomyces griseus (WO11/150157) and S. pristinaespiralis(WO12/137147). Other examples are lipase polypeptides such as thosedescribed in EP407225, WO92/05249, WO94/01541, WO94/25578, WO95/14783,WO95/30744, WO95/35381, WO95/22615, WO96/00292, WO97/04079, WO97/07202,WO00/34450, WO00/60063, WO01/92502, WO07/87508 and WO09/109500.Preferred commercial lipase products include Lipolase™, Lipex™; Lipolex™and Lipoclean™ (Novozymes NS), Lumafast (originally from Genencor) andLipomax (originally from Gist-Brocades). Yet other examples are lipasessometimes referred to as acyltransferases or perhydrolases, e.g.acyltransferases with homology to Candida antarctica lipase A(WO10/111143), acyltransferase from Mycobacterium smegmatis(WO05/56782), perhydrolases from the CE 7 family (WO09/67279), andpolypeptides of the M. smegmatis perhydrolase in particular the S54Vvariant used in the commercial product Gentle Power Bleach from HuntsmanTextile Effects Pte Ltd (WO10/100028).

In addition to the polypeptides having hexosaminidase activitycomprising SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ IDNO 11 or a polypeptide having hexosaminidase activity and having atleast 60% sequence identity hereto the cleaning composition of theinvention may further comprise amylases which can be used together witha polypeptide of the invention. The amylase may be an alpha-amylase or aglucoamylase and may be of bacterial or fungal origin. Chemicallymodified or protein engineered mutants are included. Amylases include,for example, alpha-amylases obtained from Bacillus, e.g., a specialstrain of Bacillus licheniformis, described in more detail in GB1,296,839. Suitable amylases include amylases having SEQ ID NO 3 in WO95/10603 or polypeptides having 90% sequence identity to SEQ ID NO 3thereof. Preferred polypeptides are described in WO 94/02597, WO94/18314, WO 97/43424 and SEQ ID NO 4 of WO 99/019467, such aspolypeptides with substitutions in one or more of the followingpositions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181,188, 190, 197, 201, 202, 207, 208, 209, 211, 243, 264, 304, 305, 391,408, and 444. Different suitable amylases include amylases having SEQ IDNO 6 in WO 02/010355 or polypeptides thereof having 90% sequenceidentity to SEQ ID NO 6. Preferred polypeptides of SEQ ID NO 6 are thosehaving a deletion in positions 181 and 182 and a substitution inposition 193. Other amylases which are suitable are hybrid alpha-amylasecomprising residues 1-33 of the alpha-amylase derived from B.amyloliquefaciens shown in SEQ ID NO 6 of WO 2006/066594 and residues36-483 of the B. licheniformis alpha-amylase shown in SEQ ID NO 4 of WO2006/066594 or polypeptides having 90% sequence identity thereof.Preferred polypeptides of this hybrid alpha-amylase are those having asubstitution, a deletion or an insertion in one of more of the followingpositions: G48, T49, G107, H156, A181, N190, M197, I201, A209 and Q264.Most preferred polypeptides of the hybrid alpha-amylase comprisingresidues 1-33 of the alpha-amylase derived from B. amyloliquefaciensshown in SEQ ID NO 6 of WO 2006/066594 and residues 36-483 of SEQ ID NO4 are those having the substitutions:

M197T;

H156Y+A181T+N190F+A209V+Q264S; or

G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S.

Further amylases which are suitable are amylases having SEQ ID NO 6 inWO 99/019467 or polypeptides thereof having 90% sequence identity to SEQID NO 6. Preferred polypeptides of SEQ ID NO 6 are those having asubstitution, a deletion or an insertion in one or more of the followingpositions: R181, G182, H183, G184, N195, I206, E212, E216 and K269.Particularly preferred amylases are those having deletion in positionsR181 and G182, or positions H183 and G184. Additional amylases which canbe used are those having SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 2 or SEQ IDNO 7 of WO 96/023873 or polypeptides thereof having 90% sequenceidentity to SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 or SEQ ID NO 7.Preferred polypeptides of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 or SEQID NO 7 are those having a substitution, a deletion or an insertion inone or more of the following positions: 140, 181, 182, 183, 184, 195,206, 212, 243, 260, 269, 304 and 476. More preferred polypeptides arethose having a deletion in positions 181 and 182 or positions 183 and184. Most preferred amylase polypeptides of SEQ ID NO 1, SEQ ID NO 2 orSEQ ID NO 7 are those having a deletion in positions 183 and 184 and asubstitution in one or more of positions 140, 195, 206, 243, 260, 304and 476. Other amylases which can be used are amylases having SEQ ID NO2 of WO 08/153815, SEQ ID NO 10 in WO 01/66712 or polypeptides thereofhaving 90% sequence identity to SEQ ID NO 2 of WO 08/153815 or 90%sequence identity to SEQ ID NO 10 in WO 01/66712. Preferred polypeptidesof SEQ ID NO 10 in WO 01/66712 are those having a substitution, adeletion or an insertion in one of more of the following positions: 176,177, 178, 179, 190, 201, 207, 211 and 264. Further suitable amylases areamylases having SEQ ID NO 2 of WO 09/061380 or polypeptides having 90%sequence identity to SEQ ID NO 2 thereof. Preferred polypeptides of SEQID NO 2 are those having a truncation of the C-terminus and/or asubstitution, a deletion or an insertion in one of more of the followingpositions: Q87, Q98, S125, N128, T131, T165, K178, R180, S181, T182,G183, M201, F202, N225, S243, N272, N282, Y305, R309, D319, Q320, Q359,K444 and G475. More preferred polypeptides of SEQ ID NO 2 are thosehaving the substitution in one of more of the following positions:Q87E,R, Q98R, S125A, N128C, T131I, T165I, K178L, T182G, M201L, F202Y,N225E,R, N272E,R, S243Q,A,E,D, Y305R, R309A, Q320R, Q359E, K444E andG475K and/or deletion in position R180 and/or S181 or of T182 and/orG183. Most preferred amylase polypeptides of SEQ ID NO 2 are thosehaving the substitutions:

N128C+K178L+T182G+Y305R+G475K;

N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;

S125A+N128C+K178L+T182G+Y305R+G475K; or

S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K wherein the polypeptidesare C-terminally truncated and optionally further comprises asubstitution at position 243 and/or a deletion at position 180 and/orposition 181.

Other suitable amylases are the alpha-amylase having SEQ ID NO 12 inWO01/66712 or a variant having at least 90% sequence identity to SEQ IDNO 12. Preferred amylase polypeptides are those having a substitution, adeletion or an insertion in one of more of the following positions ofSEQ ID NO 12 in WO01/66712: R28, R118, N174; R181, G182, D183, G184,G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320,H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484.Particular preferred amylases include polypeptides having a deletion ofD183 and G184 and having the substitutions R118K, N195F, R320K andR458K, and a variant additionally having substitutions in one or moreposition selected from the group: M9, G149, G182, G186, M202, T257,Y295, N299, M323, E345 and A339, most preferred a variant thatadditionally has substitutions in all these positions.

Other examples are amylase polypeptides such as those described inWO2011/098531, WO2013/001078 and WO2013/001087.

Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™,Stainzyme™, Stainzyme Plus™, Natalase™, Liquozyme X and BAN™ (fromNovozymes NS), and Rapidase™, Purastar™/Effectenz™, Powerase andPreferenz S100 (from Genencor International Inc./DuPont).

In addition to the polypeptides having hexosaminidase activitycomprising the polypeptides of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, ora polypeptide having hexosaminidase activity and having at least 60%sequence identity hereto the cleaning composition of the invention mayfurther comprise peroxidases/oxidases including those of plant,bacterial or fungal origin. Chemically modified or protein engineeredmutants are included. Examples of useful peroxidases include peroxidasesfrom Coprinus, e.g., from C. cinereus, and polypeptides thereof as thosedescribed in WO 93/24618, WO 95/10602, and WO 98/15257. Commerciallyavailable peroxidases include Guardzyme™ (Novozymes NS).

The cleaning composition enzyme(s) may be included in e.g. a detergentcomposition by adding separate additives containing one or more enzymes,or by adding a combined additive comprising these enzymes. A detergentadditive of the invention, i.e., a separate additive or a combinedadditive, can be formulated, for example, as a granulate, liquid,slurry, etc. Preferred detergent additive formulations are granulates,in particular non-dusting granulates, liquids, in particular stabilizedliquids, or slurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238,216.

The detergent compositions of the invention may also contain 0-10% byweight, such as 0.5-5%, 2-5%, 0.5-2% or 0.2-1% of a polymer. Any polymerknown in the art for use in detergents may be utilized. The polymer mayfunction as a co-builder as mentioned above, or may provideantiredeposition, fiber protection, soil release, dye transferinhibition, grease cleaning and/or anti-foaming properties. Somepolymers may have more than one of the above-mentioned properties and/ormore than one of the below-mentioned motifs. Exemplary polymers include(carboxymethyl)cellulose (CMC), poly(vinyl alcohol) (PVA),poly(vinylpyrrolidone) (PVP), poly(ethyleneglycol) or poly(ethyleneoxide) (PEG), ethoxylated poly(ethyleneimine), (carboxymethyl)inulin(CMI), and polycarboxylates such as PAA, PAA/PMA, polyaspartic acid, andlauryl methacrylate/acrylic acid copolymers, hydrophobically modifiedCMC (HM-CMC) and silicones, copolymers of terephthalic acid andoligomeric glycols, copolymers of poly(ethylene terephthalate) andpoly(oxyethene terephthalate) (PET-POET), PVP, poly(vinylimidazole)(PVI), poly(vinylpyridine-N-oxide) (PVPO or PVPNO) andpolyvinylpyrrolidone-vinylimidazole (PVPVI). Further exemplary polymersinclude sulfonated polycarboxylates, polyethylene oxide andpolypropylene oxide (PEO-PPO) and diquaternium ethoxy sulfate. Otherexemplary polymers are disclosed in, e.g., WO 2006/130575. Salts of theabove-mentioned polymers are also contemplated.

The cleaning compositions of the present invention may also includefabric hueing agents such as dyes or pigments, which when formulated indetergent compositions can deposit onto a fabric when said fabric iscontacted with a wash liquor comprising said detergent compositions andthus altering the tint of said fabric through absorption/reflection ofvisible light. Fluorescent whitening agents emit at least some visiblelight if subjected to ultraviolet light. In contrast, fabric hueingagents alter the tint of a surface as they absorb at least a portion ofthe visible light spectrum. Suitable fabric hueing agents include dyesand dye-clay conjugates, and may also include pigments. Suitable dyesinclude small molecule dyes and polymeric dyes. Suitable small moleculedyes include small molecule dyes selected from the group consisting ofdyes falling into the Colour Index (C.I.) classifications of DirectBlue, Direct Red, Direct Violet, Acid Blue, Acid Red, Acid Violet, BasicBlue, Basic Violet and Basic Red, or mixtures thereof, for example asdescribed in WO2005/03274, WO2005/03275, WO2005/03276 and EP1876226(hereby incorporated by reference). The detergent composition preferablycomprises from about 0.00003 wt % to about 0.2 wt %, from about 0.00008wt % to about 0.05 wt %, or even from about 0.0001 wt % to about 0.04 wt% fabric hueing agent. The composition may comprise from 0.0001 wt % to0.2 wt % fabric hueing agent, this may be especially preferred when thecomposition is in the form of a unit dose pouch. Suitable hueing agentsare also disclosed in, e.g. WO 2007/087257 and WO2007/087243.

The composition may further contain 0-10% by weight, for example 0-5% byweight, such as about 0.5 to about 5%, or about 3% to about 5%, of ahydrotrope. Any hydrotrope known in the art for use in detergents may beutilized. Non-limiting examples of hydrotropes include sodiumbenzenesulfonate, sodium p-toluene sulfonate (STS), sodium xylenesulfonate (SXS), sodium cumene sulfonate (SCS), sodium cymene sulfonate,amine oxides, alcohols and polyglycolethers, sodium hydroxynaphthoate,sodium hydroxynaphthalene sulfonate, sodium ethylhexyl sulfate, andcombinations thereof.

The cleaning compositions of the present invention can also containdispersants. In particular, powdered detergents may comprisedispersants. Suitable water-soluble organic materials include the homo-or co-polymeric acids or their salts, in which the polycarboxylic acidcomprises at least two carboxyl radicals separated from each other bynot more than two carbon atoms. Suitable dispersants are for exampledescribed in Powdered Detergents, Surfactant science series volume 71,Marcel Dekker, Inc.

The cleaning composition may also include one or more dye transferinhibiting agents. Suitable polymeric dye transfer inhibiting agentsinclude, but are not limited to, polyvinylpyrrolidone polymers,polyamine-N-oxide polymers, copolymers of N-vinylpyrrolidone andN-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles ormixtures thereof. When present in a subject composition, the dyetransfer inhibiting agents may be present at levels from about 0.0001%to about 10%, from about 0.01% to about 5% or even from about 0.1% toabout 3% by weight of the composition.

The cleaning compositions may preferably also contain additionalcomponents that may tint articles being cleaned, such as fluorescentwhitening agent or optical brighteners. Where present the brightener ispreferably at a level of about 0.01% to about 0.5%. Any fluorescentwhitening agent suitable for use in a laundry detergent composition maybe used in a laundry composition. The most commonly used fluorescentwhitening agents are those belonging to the classes ofdiaminostilbene-sulfonic acid derivatives, diarylpyrazoline derivativesand biphenyl-distyryl derivatives. Examples of thediaminostilbene-sulfonic acid derivative type of fluorescent whiteningagents include the sodium salts of:4,4′-bis[(4-anilino-6-diethanolamino-s-triazin-2-yl)amino]stilbene-2,2′-disulfonate,4,4′-bis[(4,6-dianilino-s-triazin-2-yl)amino]stilbene-2,2′-disulfonate,4,4′-bis{4-anilino-6-[methyl(2-hydroxyethyl)amino]-s-triazin-2-ylamino}stilbene-2,2′-disulfonate,4,4′-bis(4-phenyl-1,2,3-triazol-2-yl)stilbene-2,2′-disulfonate andsodium5-(2H-naphtho[1,2-d][1,2,3]triazol-2-yl)-2-[(E)-2-phenylvinyl]benzenesulfonate.Preferred fluorescent whitening agents are Tinopal DMS and Tinopal CBSavailable from BASF. Tinopal DMS is the disodium salt of4,4′-bis[(4-anilino-6-morpholino-s-triazin-2-yl)amino]stilbene-2,2′-disulfonate.Tinopal CBS is the disodium salt of2,2′-[biphenyl-4,4′-di(2,1-ethenediyl)]dibenzene-1-sulfonate. Alsopreferred is the commercially available Parawhite KX, supplied byParamount Minerals and Chemicals, Mumbai, India. Other fluorescerssuitable for use include the 1-3-diarylpyrazolines and the7-alkylaminocoumarins.

Suitable fluorescent brightener levels include lower levels of fromabout 0.01, from 0.05, from about 0.1 or even from about 0.2 wt % toupper levels of 0.5 or even 0.75 wt %.

The cleaning composition may also include one or more soil-releasepolymers which aid the removal of soils from fabrics such as cotton andpolyester-based fabrics, in particular the removal of hydrophobic soilsfrom polyester-based fabrics. The soil release polymers may for examplebe nonionic or anionic terephthalate-based polymers,polyvinylcaprolactam and related copolymers, vinyl graft copolymers orpolyester polyamides; see for example Chapter 7 in Powdered Detergents,Surfactant science series volume 71, Marcel Dekker, Inc. Another type ofsoil release polymers is amphiphilic alkoxylated grease cleaningpolymers comprising a core structure and a plurality of alkoxylategroups attached to that core structure. The core structure may comprisea polyalkylenimine structure or a polyalkanolamine structure asdescribed in detail in WO 2009/087523 (hereby incorporated byreference). Furthermore, random graft co-polymers are suitablesoil-release polymers. Suitable graft co-polymers are described in moredetail in WO 2007/138054, WO 2006/108856 and WO 2006/113314 (herebyincorporated by reference). Other soil-release polymers are substitutedpolysaccharide structures especially substituted cellulosic structuressuch as modified cellulose derivatives such as those described in EP1867808 or WO 2003/040279 (both are hereby incorporated by reference).Suitable cellulosic polymers include cellulose, cellulose ethers,cellulose esters, cellulose amides and mixtures thereof. Suitablecellulosic polymers include anionically modified cellulose, nonionicallymodified cellulose, cationically modified cellulose, zwitterionicallymodified cellulose, and mixtures thereof.

The cleaning compositions may also include one or more anti-redepositionagents such as (carboxymethyl) cellulose (CMC), poly(vinyl alcohol)(PVA), homopolymers of acrylic acid, copolymers of acrylic acid andmaleic acid, and ethoxylated polyethyleneimines. The cellulose basedpolymers described under soil-release polymers above may also functionas anti-redeposition agents.

The cleaning composition may also contain one are more adjunct material.Suitable adjunct materials include, but are not limited to, anti-shrinkagents, anti-wrinkling agents, bactericides, binders, carriers, dyes,enzyme stabilizers, fabric softeners, fillers, foam regulators,hydrotropes, perfumes, pigments, sod suppressors, solvents, andstructurants for liquid detergents and/or structure elasticizing agents.

The cleaning composition may be in any convenient form, e.g., a bar, ahomogenous tablet, a tablet having two or more layers, a pouch havingone or more compartments, a regular or compact powder, a granule, apaste, a gel, or a regular, compact or concentrated liquid.

Pouches can be configured as single or multicompartments. It can be ofany form, shape and material which is suitable for hold the composition,e.g. without allowing the release of the composition to release of thecomposition from the pouch prior to water contact. The pouch is madefrom water soluble film which encloses an inner volume. Said innervolume can be divided into compartments of the pouch. Preferred filmsare polymeric materials preferably polymers which are formed into a filmor sheet. Preferred polymers, copolymers or derivates thereof areselected polyacrylates, and water soluble acrylate copolymers, methylcellulose, carboxy methyl cellulose, sodium dextrin, ethyl cellulose,hydroxyethyl cellulose, hydroxypropyl methyl cellulose, malto dextrin,poly methacrylates, most preferably polyvinyl alcohol copolymers and,hydroxypropyl methyl cellulose (HPMC). Preferably the level of polymerin the film for example PVA is at least about 60%. Preferred averagemolecular weight will typically be about 20,000 to about 150,000. Filmscan also be of blended compositions comprising hydrolytically degradableand water soluble polymer blends such as polylactide and polyvinylalcohol (known under the Trade reference M8630 as sold by MonoSol LLC,Indiana, USA) plus plasticisers like glycerol, ethylene glycerol,propylene glycol, sorbitol and mixtures thereof. The pouches cancomprise a solid laundry cleaning composition or part components and/ora liquid cleaning composition or part components separated by thewater-soluble film. The compartment for liquid components can bedifferent in composition than compartments containing solids:US2009/0011970 A1.

Detergent ingredients can be separated physically from each other bycompartments in water dissolvable pouches or in different layers oftablets. Thereby negative storage interaction between components can beavoided. Different dissolution profiles of each of the compartments canalso give rise to delayed dissolution of selected components in the washsolution.

A liquid or gel detergent, which is not unit dosed, may be aqueous,typically containing at least 20% by weight and up to 95% water, such asup to about 70% water, up to about 65% water, up to about 55% water, upto about 45% water, up to about 35% water. Other types of liquids,including without limitation, alkanols, amines, diols, ethers andpolyols may be included in an aqueous liquid or gel. An aqueous liquidor gel detergent may contain from 0-30% organic solvent.

The present invention is also directed to methods for using thepolypeptides according to the invention or compositions thereof inlaundering of textile and fabrics, such as house hold laundry washingand industrial laundry washing.

One aspect of the invention relates to a method for laundering an itemcomprising:

a) expose of an item to a wash liquor comprising a polypeptide selectedfrom the group consisting of polypeptides shown in SEQ ID NO 7, SEQ IDNO 8, SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11 or expose of an item toa detergent composition according to any of claims 1 to 14;

b) completing at least one wash cycle; and

-   -   optionally rinsing the item, wherein the item is a textile.        The invention is also directed to methods for using the        polypeptides according to the invention or compositions thereof        in cleaning hard surfaces such as floors, tables, walls, roofs        etc. as well as surfaces of hard objects such as cars (car wash)        and dishes (dish wash).

The polypeptides of the present invention may be added to and thusbecome a component of a cleaning composition. One aspect of theinvention relates to the use of a polypeptide having hexosaminidaseactivity optionally comprising one or more motif(s) selected from thegroup consisting of GXDE (SEQ ID NO 18),[EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19),[VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO: 20), WND[SQR][IVL][TLVM] (SEQ IDNO: 21), QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO: 23), NLD[DR]S (SEQ IDNO: 24), wherein the polypeptide is selected from the group consistingof SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 andpolypeptides having at least 60% sequence identity hereto havinghexosaminidase activity in a cleaning process such as laundering and/orhard surface cleaning.

Thus, one aspect of the invention relates to the use of the use of apolypeptide having hexosaminidase activity, optionally comprising one ormore motif(s) selected from the group consisting of GXDE (SEQ ID NO 18),[EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19),[VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO: 20), WND[SQR][IVL][TLVM] (SEQ IDNO: 21), QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO: 23), NLD[DR]S (SEQ IDNO: 24), wherein the polypeptide is selected from the group consistingof SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 andpolypeptides having at least 60% sequence identity in a cleaning processsuch as laundering and/or hard surface cleaning and wherein thepolypeptide has improved deep cleaning properties, relative to areference enzyme.

The cleaning process or the textile care process may for example be alaundry process, a dishwashing process or cleaning of hard surfaces suchas bathroom tiles, floors, table tops, drains, sinks and washbasins.Laundry processes can for example be household laundering, but it mayalso be industrial laundering. Furthermore, the invention relates to aprocess for laundering of fabrics and/or garments where the processcomprises treating fabrics with a washing solution containing a cleaningcomposition, and at least one polypeptide of the invention. The cleaningprocess or a textile care process can for example be carried out in amachine washing process or in a manual washing process. The washingsolution can for example be an aqueous washing solution containing adetergent composition.

The polypeptides comprised in the Terribacillus clade are thusparticularly useful in composition comprising surfactants such asdetergent compositions and the polypeptides of the invention maypreferably be used in cleaning processes such as laundry and dish wash.

In some aspects, the present invention relates to polypeptides of theclade of Terribacillus having a sequence identity to the maturepolypeptide of SEQ ID NO 2 of at least 60%, e.g., at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100%, which havehexosaminidase activity. In one aspect, the polypeptides differ by up to10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the maturepolypeptide shown in SEQ ID NO 7.

In some aspects, the present invention relates to polypeptides of theclade of Terribacillus having a sequence identity to the maturepolypeptide of SEQ ID NO 4 of at least 60%, e.g., at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100%, which havehexosaminidase activity. In one aspect, the polypeptides differ by up to10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the maturepolypeptide shown in SEQ ID NO 8.

In some aspects, the present invention relates to polypeptides of theclade of Terribacillus having a sequence identity to the maturepolypeptide of SEQ ID NO 6 of at least 60%, e.g., at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100%, which havehexosaminidase activity. In one aspect, the polypeptides differ by up to10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the maturepolypeptide shown in SEQ ID NO 9.

In some aspects, the present invention relates to polypeptides of theclade of Terribacillus having a sequence identity to the maturepolypeptide of SEQ ID NO 13 of at least 60%, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%, which havehexosaminidase activity. In one aspect, the polypeptides differ by up to10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the maturepolypeptide shown in SEQ ID NO 10.

In some aspects, the present invention relates to polypeptides of theclade of Terribacillus having a sequence identity to the maturepolypeptide of SEQ ID NO 14 of at least 60%, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%, which havehexosaminidase activity. In one aspect, the polypeptides differ by up to10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the maturepolypeptide shown in SEQ ID NO 11.

In an embodiment, the polypeptide has been isolated. A polypeptide ofthe present invention preferably comprises or consists of the amino acidsequence of SEQ ID NO 7 or an allelic variant thereof; or is a fragmentthereof having hexosaminidase activity. In another aspect, thepolypeptide comprises or consists of the mature polypeptide of SEQ ID NO2. In another aspect, the polypeptide comprises or consists of aminoacids 1 to 324 of SEQ ID NO 2.

In an embodiment, the polypeptide has been isolated. A polypeptide ofthe present invention preferably comprises or consists of the amino acidsequence of SEQ ID NO 8 or an allelic variant thereof; or is a fragmentthereof having hexosaminidase activity. In another aspect, thepolypeptide comprises or consists of the mature polypeptide of SEQ ID NO4. In another aspect, the polypeptide comprises or consists of aminoacids 1 to 324 of SEQ ID NO 4.

In an embodiment, the polypeptide has been isolated. A polypeptide ofthe present invention preferably comprises or consists of the amino acidsequence of SEQ ID NO 9 or an allelic variant thereof; or is a fragmentthereof having hexosaminidase activity. In another aspect, thepolypeptide comprises or consists of the mature polypeptide of SEQ ID NO6. In another aspect, the polypeptide comprises or consists of aminoacids 1 to 324 of SEQ ID NO 6.

In an embodiment, the polypeptide has been isolated. A polypeptide ofthe present invention preferably comprises or consists of the amino acidsequence of SEQ ID NO 10 or an allelic variant thereof; or is a fragmentthereof having hexosaminidase activity. In another aspect, thepolypeptide comprises or consists of the mature polypeptide of SEQ ID NO13. In another aspect, the polypeptide comprises or consists of aminoacids 1 to 324 of SEQ ID NO 13.

In an embodiment, the polypeptide has been isolated. A polypeptide ofthe present invention preferably comprises or consists of the amino acidsequence of SEQ ID NO 11 or an allelic variant thereof; or is a fragmentthereof having hexosaminidase activity. In another aspect, thepolypeptide comprises or consists of the mature polypeptide of SEQ ID NO15. In another aspect, the polypeptide comprises or consists of aminoacids 1 to 324 of SEQ ID NO 15.

In another embodiment, the present invention relates to a polypeptidehaving hexosaminidase activity encoded by a polynucleotide thathybridizes under very low stringency conditions, low stringencyconditions, medium stringency conditions, medium-high stringencyconditions, high stringency conditions, or very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO1, 3, 5, 12 or 14 or (ii) the full-length complement of (i) (Sambrook etal., 1989, Molecular Cloning, A Laboratory Manual, 2d edition, ColdSpring Harbor, New York). In an embodiment, the polypeptide has beenisolated.

The polynucleotide of SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO12 or SEQ ID NO 14 or a subsequence thereof, as well as the maturepolypeptide of SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 13, SEQID NO 15 or a fragment thereof may be used to design nucleic acid probesto identify and clone DNA encoding polypeptides having hexosaminidaseactivity from strains of different genera or species according tomethods well known in the art. Such probes can be used for hybridizationwith the genomic DNA or cDNA of a cell of interest, following standardSouthern blotting procedures, to identify and isolate the correspondinggene therein. Such probes can be considerably shorter than the entiresequence, but should be at least 15, e.g., at least 25, at least 35, orat least 70 nucleotides in length. Preferably, the nucleic acid probe isat least 100 nucleotides in length, e.g., at least 200 nucleotides, atleast 300 nucleotides, at least 400 nucleotides, at least 500nucleotides, at least 600 nucleotides, at least 700 nucleotides, atleast 800 nucleotides, or at least 900 nucleotides in length. Both DNAand RNA probes can be used. The probes are typically labeled fordetecting the corresponding gene (for example, with ³²P, ³H, ³⁵S,biotin, or avidin). Such probes are encompassed by the presentinvention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a polypeptide having hexosaminidase activity. Genomic or otherDNA from such other strains may be separated by agarose orpolyacrylamide gel electrophoresis, or other separation techniques. DNAfrom the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Toidentify a clone or DNA that hybridizes with SEQ ID NO 1, 3, 5, 12 or 14or a subsequence thereof, the carrier material is used in a Southernblot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 12 or SEQ ID NO14; (ii) the mature polypeptide coding sequence of SEQ ID NO 1, SEQ IDNO 3, SEQ ID NO 5, SEQ ID NO 12 or SEQ ID NO 14; (iii) the full-lengthcomplement thereof; or (iv) a subsequence thereof; under very low tovery high stringency conditions. Molecules to which the nucleic acidprobe hybridizes under these conditions can be detected using, forexample, X-ray film or any other detection means known in the art.

In another embodiment, the present invention relates to a polypeptidehaving hexosaminidase activity encoded by a polynucleotide having asequence identity to the mature polypeptide coding sequence of SEQ ID NO1 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%. In a further embodiment, thepolypeptide has been isolated.

In another embodiment, the present invention relates to a polypeptidehaving hexosaminidase activity encoded by a polynucleotide having asequence identity to the mature polypeptide coding sequence of SEQ ID NO3 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%. In a further embodiment, thepolypeptide has been isolated.

In another embodiment, the present invention relates to a polypeptidehaving hexosaminidase activity encoded by a polynucleotide having asequence identity to the mature polypeptide coding sequence of SEQ ID NO5 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%. In a further embodiment, thepolypeptide has been isolated.

In another embodiment, the present invention relates to a polypeptidehaving hexosaminidase activity encoded by a polynucleotide having asequence identity to the mature polypeptide coding sequence of SEQ ID NO12 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%. In a further embodiment, thepolypeptide has been isolated.

In another embodiment, the present invention relates to a polypeptidehaving hexosaminidase activity encoded by a polynucleotide having asequence identity to the mature polypeptide coding sequence of SEQ ID NO14 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%. In a further embodiment, thepolypeptide has been isolated.

In another embodiment, the present invention relates to variants of themature polypeptide of SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO13 or SEQ ID NO 15 comprising a substitution, deletion, and/or insertionat one or more (e.g., several) positions. In an embodiment, the numberof amino acid substitutions, deletions and/or insertions introduced intothe mature polypeptide of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ IDNO 10 or SEQ ID NO 11 is up to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or10. The amino acid changes may be of a minor nature, that isconservative amino acid substitutions or insertions that do notsignificantly affect the folding and/or activity of the protein; smalldeletions, typically of 1-30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to 20-25 residues; or a smallextension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain. Examples of conservative substitutions are withinthe groups of basic amino acids (arginine, lysine and histidine), acidicamino acids (glutamic acid and aspartic acid), polar amino acids(glutamine and asparagine), hydrophobic amino acids (leucine, isoleucineand valine), aromatic amino acids (phenylalanine, tryptophan andtyrosine), and small amino acids (glycine, alanine, serine, threonineand methionine). Amino acid substitutions that do not generally alterspecific activity are known in the art and are described, for example,by H. Neurath and R. L. Hill, 1979, In, The Proteins, Academic Press,New York. Common substitutions are Ala/Ser, Asp/Glu, Thr/Ser, Ala/Gly,Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn,Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like. Essential amino acids in a polypeptide can be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant molecules are tested for hexosaminidase activity to identifyamino acid residues that are critical to the activity of the molecule.See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The activesite of the enzyme or other biological interaction can also bedetermined by physical analysis of structure, as determined by suchtechniques as nuclear magnetic resonance, crystallography, electrondiffraction, or photoaffinity labeling, in conjunction with mutation ofputative contact site amino acids. See, for example, de Vos et al.,1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity ofessential amino acids can also be inferred from an alignment with arelated polypeptide. The polypeptides of the invention arehexosaminidase e.g. dispersins belonging to the Terribacillus clade.Terribacillus dispersins are β-hexosaminidases that specificallyhydrolyzes β-1,6-glycosidic linkages of N-acetylglucosamine polymers,which may be found e.g. in biofilm. Dispersin B which belongs to adifferent clade than the polypeptides (hexosaminidases) of the inventioncontains three highly conserved acidic residues: an aspartic acid atresidue 183 (D183), a glutamic acid at residue 184 (E184), and aglutamic acid at residue 332 (E332). Single or multiple amino acidsubstitutions, deletions, and/or insertions can be made and tested usingknown methods of mutagenesis, recombination, and/or shuffling, followedby a relevant screening procedure, such as those disclosed byReidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer,1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO95/22625. Other methods that can be used include error-prone PCR, phageDsplay (e.g., Lowman et al., 1991, Biochemistry 30: 10832-10837; U.S.Pat. No. 5,223,409; WO 92/06204), and region-directed mutagenesis(Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide. The polypeptide may be a hybrid polypeptide in which aregion of one polypeptide is fused at the N-terminus or the C-terminusof a region of another polypeptide.

The polypeptide may be a fusion polypeptide or cleavable fusionpolypeptide in which another polypeptide is fused at the N-terminus orthe C-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779). A fusionpolypeptide can further comprise a cleavage site between the twopolypeptides. Upon secretion of the fusion protein, the site is cleavedreleasing the two polypeptides. Examples of cleavage sites include, butare not limited to, the sites disclosed in Martin et al., 2003, J. Ind.Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol.76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol.63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; andContreras et al., 1991, Biotechnology 9: 378-381; Eaton et al., 1986,Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13:982-987; Carter et al., 1989, Proteins: Structure, Function, andGenetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.

Sources of Polypeptides Having Hexosaminidase Activity

A polypeptide having hexosaminidase activity of the present inventionmay be obtained from microorganisms of any genus. For purposes of thepresent invention, the term “obtained from” as used herein in connectionwith a given source shall mean that the polypeptide encoded by apolynucleotide is produced by the source or by a strain in which thepolynucleotide from the source has been inserted. In one aspect, thepolypeptide obtained from a given source is secreted extracellularly.

In another aspect, the polypeptide is a Terribacillus polypeptide, e.g.,a polypeptide obtained from Terribacillus saccharophilus. In a preferredaspect, the polypeptide is a polypeptide having at least 80% sequenceidentity to SEQ ID NO 7 and is obtained from Terribacillus preferablyTerribacillus saccharophilus.

In another aspect, the polypeptide is a Terribacillus polypeptide, e.g.,a polypeptide obtained from Terribacillus goriensis. In a preferredaspect, the polypeptide is a polypeptide having at least 80% sequenceidentity to SEQ ID NO 8 and is obtained from Terribacillus preferablyTerribacillus goriensis.

In another aspect, the polypeptide is a Terribacillus polypeptide, e.g.,a polypeptide obtained from Terribacillus saccharophilus. In a preferredaspect, the polypeptide is a polypeptide having at least 80% sequenceidentity to SEQ ID NO 9 and is obtained from Terribacillus preferablyTerribacillus saccharophilus.

In another aspect, the polypeptide is a Terribacillus polypeptide, e.g.,a polypeptide obtained from Terribacillus saccharophilus. In a preferredaspect, the polypeptide is a polypeptide having at least 80% sequenceidentity to SEQ ID NO 10 and is obtained from Terribacillus preferablyTerribacillus saccharophilus.

In another aspect, the polypeptide is a Terribacillus polypeptide, e.g.,a polypeptide obtained from Terribacillus saccharophilus. In a preferredaspect, the polypeptide is a polypeptide having at least 80% sequenceidentity to SEQ ID NO 11 and is obtained from Terribacillus preferablyTerribacillus saccharophilus.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The polypeptide may be identified and obtained from other sourcesincluding microorganisms isolated from nature (e.g., soil, composts,water, etc.) or DNA samples obtained directly from natural materials(e.g., soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms and DNA directly from naturalhabitats are well known in the art. A polynucleotide encoding thepolypeptide may then be obtained by similarly screening a genomic DNA orcDNA library of another microorganism or mixed DNA sample. Once apolynucleotide encoding a polypeptide has been detected with theprobe(s), the polynucleotide can be isolated or cloned by utilizingtechniques that are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the expression of the coding sequence in asuitable host cell under conditions compatible with the controlsequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of the polypeptide. Manipulation of the polynucleotideprior to its insertion into a vector may be desirable or necessarydepending on the expression vector. The techniques for modifyingpolynucleotides utilizing recombinant DNA methods are well known in theart.

The control sequence may be a promoter, a polynucleotide that isrecognized by a host cell for expression of a polynucleotide encoding apolypeptide of the present invention. The promoter containstranscriptional control sequences that mediate the expression of thepolypeptide. The promoter may be any polynucleotide that showstranscriptional activity in the host cell including variant, truncated,and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xyIA and xyIB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIll, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor, as well as the NA2-tpi promoter (a modified promoterfrom an Aspergillus neutral alpha-amylase gene in which the untranslatedleader has been replaced by an untranslated leader from an Aspergillustriose phosphate isomerase gene; non-limiting examples include modifiedpromoters from an Aspergillus niger neutral alpha-amylase gene in whichthe untranslated leader has been replaced by an untranslated leader froman Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerasegene); and variant, truncated, and hybrid promoters thereof. Otherpromoters are described in U.S. Pat. No. 6,011,147.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminator isoperably linked to the 3′-terminus of the polynucleotide encoding thepolypeptide. Any terminator that is functional in the host cell may beused in the present invention.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans acetamidase, Aspergillusnidulans anthranilate synthase, Aspergillus niger glucoamylase,Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase,Fusarium oxysporum trypsin-like protease, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor. Preferred terminators for yeast host cells areobtained from the genes for Saccharomyces cerevisiae enolase,Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomycescerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other usefulterminators for yeast host cells are described by Romanos et al., 1992,supra. The control sequence may also be an mRNA stabilizer regiondownstream of a promoter and upstream of the coding sequence of a genewhich increases expression of the gene. Examples of suitable mRNAstabilizer regions are obtained from a Bacillus thuringiensis cryIIIAgene (WO 94/25612) and a Bacillus subtilis SP82 gene (Hue et al., 1995,Journal of Bacteriology 177: 3465-3471). The control sequence may alsobe a leader, a nontranslated region of an mRNA that is important fortranslation by the host cell. The leader is operably linked to the5′-terminus of the polynucleotide encoding the polypeptide. Any leaderthat is functional in the host cell may be used. Preferred leaders forfilamentous fungal host cells are obtained from the genes forAspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase. Suitable leaders for yeast host cells are obtainedfrom the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomycescerevisiae alpha-factor, and Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP). Thecontrol sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used. Preferredpolyadenylation sequences for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidaseAspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease. Useful polyadenylation sequences for yeast host cells aredescribed by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. A foreign signal peptide coding sequence may be required wherethe coding sequence does not naturally contain a signal peptide codingsequence. Alternatively, a foreign signal peptide coding sequence maysimply replace the natural signal peptide coding sequence in order toenhance secretion of the polypeptide. However, any signal peptide codingsequence that directs the expressed polypeptide into the secretorypathway of a host cell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137. Effective signal peptide coding sequences for filamentousfungal host cells are the signal peptide coding sequences obtained fromthe genes for Aspergillus niger neutral amylase, Aspergillus nigerglucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolenscellulase, Humicola insolens endoglucanase V, Humicola lanuginosalipase, and Rhizomucor miehei aspartic proteinase. Useful signalpeptides for yeast host cells are obtained from the genes forSaccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of apolypeptide and the signal peptide sequence is positioned next to theN-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the polypeptide relative to the growth of the host cell.Examples of regulatory sequences are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysequences in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter,and Trichoderma reesei cellobiohydrolase II promoter may be used. Otherexamples of regulatory sequences are those that allow for geneamplification. In eukaryotic systems, these regulatory sequences includethe dihydrofolate reductase gene that is amplified in the presence ofmethotrexate, and the metallothionein genes that are amplified withheavy metals. In these cases, the polynucleotide encoding thepolypeptide would be operably linked to the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleotideand control sequences may be joined together to produce a recombinantexpression vector that may include one or more convenient restrictionsites to allow for insertion or substitution of the polynucleotideencoding the polypeptide at such sites. Alternatively, thepolynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin, or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, adeA(phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB(phosphoribosyl-aminoimidazole synthase), amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene. Preferred for use in a Trichodermacell are adeA, adeB, amdS, hph, and pyrG genes.

The selectable marker may be a dual selectable marker system asdescribed in WO 2010/039889. In one aspect, the dual selectable markeris an hph-tk dual selectable marker system.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMR1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a polypeptide. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the production of a polypeptide of thepresent invention. A construct or vector comprising a polynucleotide isintroduced into a host cell so that the construct or vector ismaintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thepolypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma. Thebacterial host cell may be any Bacillus cell including, but not limitedto, Bacillus alkalophilus, Bacillus altitudinis, Bacillusamyloliquefaciens, B. amyloliquefaciens subsp. plantarum, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus methylotrophicus, Bacilluspumilus, Bacillus safensis, Bacillus stearothermophilus, Bacillussubtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell including, butnot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397) or conjugation (see, e.g.,Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptideof the present invention, comprising (a) cultivating a cell, which inits wild-type form produces the polypeptide, under conditions conducivefor production of the polypeptide; and optionally, (b) recovering thepolypeptide. In one aspect, the cell is a Terribacillus cell. In anotheraspect, the cell is a Terribacillus saccharophilus cell. In one aspect,the cell is a Terribacillus cell. In another aspect, the cell is aTerribacillus goriensis cell.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising (a) cultivating a recombinant hostcell of the present invention under conditions conducive for productionof the polypeptide; and optionally, (b) recovering the polypeptide.

The host cells are cultivated in a nutrient medium suitable forproduction of the polypeptide using methods known in the art. Forexample, the cells may be cultivated by shake flask cultivation, orsmall-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptides having hexosaminidase activity. Thesedetection methods include, but are not limited to, use of specificantibodies, formation of an enzyme product, or disappearance of anenzyme substrate. For example, an enzyme assay may be used to determinethe activity of the polypeptide.

The polypeptide may be recovered using methods known in the art. Forexample, the polypeptide may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation. In one aspect, a fermentation broth comprising thepolypeptide is recovered.

The polypeptide may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure polypeptides.

In an alternative aspect, the polypeptide is not recovered, but rather ahost cell of the present invention expressing the polypeptide is used asa source of the polypeptide.

Formulation of Detergent Products

The cleaning composition may be in any convenient form, e.g., a bar, ahomogenous tablet, a tablet having two or more layers, a pouch havingone or more compartments, a regular or compact powder, a granule, apaste, a gel, or a regular, compact or concentrated liquid. Pouches canbe configured as single or multicompartments. It can be of any form,shape and material which is suitable for hold the composition, e.g.without allowing the release of the composition to release of thecomposition from the pouch prior to water contact. The pouch is madefrom water soluble film which encloses an inner volume. Said innervolume can be divided into compartments of the pouch. Preferred filmsare polymeric materials preferably polymers which are formed into a filmor sheet. Preferred polymers, copolymers or derivates thereof areselected polyacrylates, and water soluble acrylate copolymers, methylcellulose, carboxy methyl cellulose, sodium dextrin, ethyl cellulose,hydroxyethyl cellulose, hydroxypropyl methyl cellulose, malto dextrin,poly methacrylates, most preferably polyvinyl alcohol copolymers and,hydroxypropyl methyl cellulose (HPMC). Preferably the level of polymerin the film for example PVA is at least about 60%. Preferred averagemolecular weight will typically be about 20,000 to about 150,000. Filmscan also be of blended compositions comprising hydrolytically degradableand water soluble polymer blends such as polylactide and polyvinylalcohol (known under the Trade reference M8630 as sold by MonoSol LLC,Indiana, USA) plus plasticisers like glycerol, ethylene glycerol,propylene glycol, sorbitol and mixtures thereof. The pouches cancomprise a solid laundry cleaning composition or part components and/ora liquid cleaning composition or part components separated by the watersoluble film. The compartment for liquid components can be different incomposition than compartments containing solids: US2009/0011970 A1.

Detergent ingredients can be separated physically from each other bycompartments in water dissolvable pouches or in different layers oftablets. Thereby negative storage interaction between components can beavoided. Different dissolution profiles of each of the compartments canalso give rise to delayed dissolution of selected components in the washsolution.

A liquid or gel detergent, which is not unit dosed, may be aqueous,typically containing at least 20% by weight and up to 95% water, such asup to about 70% water, up to about 65% water, up to about 55% water, upto about 45% water, up to about 35% water. Other types of liquids,including without limitation, alkanols, amines, diols, ethers andpolyols may be included in an aqueous liquid or gel. An aqueous liquidor gel detergent may contain from 0-30% organic solvent.

A liquid or gel detergent may be non-aqueous.

Laundry Soap Bars

The polypeptides of the invention may be added to laundry soap bars andused for hand washing laundry, fabrics and/or textiles. The term laundrysoap bar includes laundry bars, soap bars, combo bars, syndet bars anddetergent bars. The types of bar usually differ in the type ofsurfactant they contain, and the term laundry soap bar includes thosecontaining soaps from fatty acids and/or synthetic soaps. The laundrysoap bar has a physical form which is solid and not a liquid, gel or apowder at room temperature. The term solid is defined as a physical formwhich does not significantly change over time, i.e. if a solid object(e.g. laundry soap bar) is placed inside a container, the solid objectdoes not change to fill the container it is placed in. The bar is asolid typically in bar form but can be in other solid shapes such asround or oval. The laundry soap bar may contain one or more additionalenzymes, protease inhibitors such as peptide aldehydes (or hydrosulfiteadduct or hemiacetal adduct), boric acid, borate, borax and/orphenylboronic acid derivatives such as 4-formylphenylboronic acid, oneor more soaps or synthetic surfactants, polyols such as glycerine, pHcontrolling compounds such as fatty acids, citric acid, acetic acidand/or formic acid, and/or a salt of a monovalent cation and an organicanion wherein the monovalent cation may be for example Na⁺, K⁺ or NH₄ ⁺and the organic anion may be for example formate, acetate, citrate orlactate such that the salt of a monovalent cation and an organic anionmay be, for example, sodium formate. The laundry soap bar may alsocontain complexing agents like EDTA and HEDP, perfumes and/or differenttype of fillers, surfactants e.g. anionic synthetic surfactants,builders, polymeric soil release agents, detergent chelators,stabilizing agents, fillers, dyes, colorants, dye transfer inhibitors,alkoxylated polycarbonates, suds suppressers, structurants, binders,leaching agents, bleaching activators, clay soil removal agents,anti-redeposition agents, polymeric dispersing agents, brighteners,fabric softeners, perfumes and/or other compounds known in the art. Thelaundry soap bar may be processed in conventional laundry soap barmaking equipment such as but not limited to: mixers, plodders, e.g a twostage vacuum plodder, extruders, cutters, logo-stampers, cooling tunnelsand wrappers. The invention is not limited to preparing the laundry soapbars by any single method. The premix may be added to the soap atdifferent stages of the process. For example, the premix containing asoap, hexosaminidase, optionally one or more additional enzymes, aprotease inhibitor, and a salt of a monovalent cation and an organicanion may be prepared and the mixture is then plodded. Thehexosaminidase and optional additional enzymes may be added at the sametime as the protease inhibitor for example in liquid form. Besides themixing step and the plodding step, the process may further comprise thesteps of milling, extruding, cutting, stamping, cooling and/or wrapping.

Formulation of Enzyme in Co-Granule

The polypeptides of the invention may be formulated as a granule forexample as a co-granule that combines one or more enzymes. Each enzymewill then be present in more granules securing a more uniformdistribution of enzymes in the detergent. This also reduces the physicalsegregation of different enzymes due to different particle sizes.Methods for producing multi-enzyme co-granulates for the detergentindustry are disclosed in the IP.com disclosure IPCOM000200739D. Anotherexample of formulation of enzymes using co-granulates are disclosed inWO 2013/188331, which relates to a detergent composition comprising (a)a multi-enzyme co-granule; (b) less than 10 wt zeolite (anhydrousbasis); and (c) less than 10 wt phosphate salt (anhydrous basis),wherein said enzyme co-granule comprises from 10 to 98 wt % moisturesink components and the composition additionally comprises from 20 to 80wt % detergent moisture sink components.

The multi-enzyme co-granule may comprise a hexosaminidase and one ormore enzymes selected from the group consisting of lipases, cellulases,xyloglucanases, perhydrolases, peroxidases, lipoxygenases, laccases,hemicellulases, proteases, cellobiose dehydrogenases, xylanases, phospholipases, esterases, cutinases, pectinases, mannanases, pectate lyases,keratinases, reductases, oxidases, phenoloxidases, ligninases,pullulanases, tannases, pentosanases, lichenases glucanases,arabinosidases, hyaluronidase, chondroitinase, amylases, and mixturesthereof.

The invention is further summarized in the following paragraphs:

-   1. Use of a polypeptide having hexosaminidase activity preferably    comprising one or more of the motif(s) GXDE (SEQ ID NO 18),    [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19),    [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO: 20), WND[SQR][IVL][TLVM]    (SEQ ID NO: 21), QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO: 23) or    NLD[DR]S (SEQ ID NO: 24) for deep cleaning of an item, wherein the    item is a textile.-   2. Use according to paragraph 1 for preventing, reducing or removing    stickiness of the item.-   3. Use according to any of paragraphs 1 or 2 for pre-treating stains    on the item.-   4. Use according to any of paragraphs 1-3 for preventing, reducing    or removing re-deposition of soil during a wash cycle.-   5. Use according to any of paragraphs 1-4 for preventing, reducing    or removing adherence of soil to the item.-   6. Use according to any of the preceding paragraphs for maintaining    or improving the whiteness of the item.-   7. Use according to any of the preceding paragraphs, wherein a    malodor is reduced or removed from the item.-   8. Use according to any of the preceding composition paragraphs,    wherein the surface is a textile surface.-   9. Use according to any of the preceding composition paragraphs,    wherein the textile is made of cotton, Cotton/Polyester, Polyester,    Polyamide, Polyacryl and/or silk.-   10. Use according to any of the preceding paragraphs, wherein the    polypeptide is a polypeptide of paragraphs 47-61-   11. A composition comprising a polypeptide having hexosaminidase    activity preferably comprising one or more of the motif(s) GXDE (SEQ    ID NO 18), [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19),    [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO: 20), WND[SQR][IVL][TLVM]    (SEQ ID NO: 21), QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO: 23) or    NLD[DR]S (SEQ ID NO: 24) and an adjunct ingredient.-   12. Composition according to paragraph 11, wherein the polypeptide    is the polypeptide of paragraphs 47-61.-   13. Composition according to any of the preceding composition    paragraphs, wherein the detergent adjunct ingredient is selected    from the group consisting of surfactants, builders, flocculating    aid, chelating agents, dye transfer inhibitors, enzymes, enzyme    stabilizers, enzyme inhibitors, catalytic materials, bleach    activators, hydrogen peroxide, sources of hydrogen peroxide,    preformed peracids, polymeric dispersing agents, clay soil    removal/anti-redeposition agents, brighteners, suds suppressors,    dyes, perfumes, structure elasticizing agents, fabric softeners,    carriers, hydrotropes, builders and co-builders, fabric huing    agents, anti-foaming agents, dispersants, processing aids, and/or    pigments.-   14. Composition according to any of the preceding composition    paragraphs wherein the composition comprises from about 5 wt % to    about 50 wt %, from about 5 wt % to about 40 wt %, from about 5 wt %    to about 30 wt %, from about 5 wt % to about 20 wt %, from about 5    wt % to about 10 wt % anionic surfactant, preferably selected from    linear alkylbenzenesulfonates (LAS), isomers of LAS, branched    alkylbenzenesulfonates (BABS), phenylalkanesulfonates,    alpha-olefinsulfonates (AOS), olefin sulfonates, alkene sulfonates,    alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonates and    disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate    (SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS),    alcohol ethersulfates (AES or AEOS or FES), secondary    alkanesulfonates (SAS), paraffin sulfonates (PS), ester sulfonates,    sulfonated fatty acid glycerol esters, alpha-sulfo fatty acid methyl    esters (alpha-SFMe or SES) including methyl ester sulfonate (MES),    alkyl- or alkenylsuccinic acid, dodecenyl/tetradecenyl succinic acid    (DTSA), fatty acid derivatives of amino acids, diesters and    monoesters of sulfo-succinic acid or salt of fatty acids (soap), and    combinations thereof.-   15. Composition according to any of the preceding composition    paragraphs wherein the composition comprises from about 10 wt % to    about 50 wt % of at least one builder, preferably selected from    citric acid, methylglycine-N, N-diacetic acid (MGDA) and/or glutamic    acid-N, N-diacetic acid (GLDA) and mixtures thereof.-   16. Composition according to any of the preceding composition    paragraphs wherein the polypeptide having hexosaminidase activity is    selected from the group consisting of polypeptides having the amino    acid sequence of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO    10, SEQ ID NO 11 and polypeptides having at least 60% e.g. 80%, 85%,    90%, 95%, 98% or 99% sequence identity hereto.-   17. Composition according to any of the preceding composition    paragraphs wherein the polypeptide having hexosaminidase activity is    the amino acid sequence of SEQ ID NO 7 or polypeptides having at    least 60% e.g. 80%, 85%, 90%, 95%, 98% or 99% sequence identity    hereto.-   18. Composition according to any of the paragraphs 11 to 16, wherein    the polypeptide having hexosaminidase activity is the amino acid    sequence of SEQ ID NO 8 or polypeptides having at least 60% e.g.    80%, 85%, 90%, 95%, 98% or 99% sequence identity hereto.-   19. Composition according to any of the paragraphs 11 to 16, wherein    the polypeptide having hexosaminidase activity is the amino acid    sequence of SEQ ID NO 9 or polypeptides having at least 60% e.g.    80%, 85%, 90%, 95%, 98% or 99% sequence identity hereto.-   20. Composition according to any of the paragraphs 11 to 16, wherein    the polypeptide having hexosaminidase activity is the amino acid    sequence of SEQ ID NO 10 or polypeptides having at least 60% e.g.    80%, 85%, 90%, 95%, 98% or 99% sequence identity hereto.-   21. Composition according to any of the paragraphs 11 to 16, wherein    the polypeptide having hexosaminidase activity is the amino acid    sequence of SEQ ID NO 11 or polypeptides having at least 60% e.g.    80%, 85%, 90%, 95%, 98% or 99% sequence identity hereto-   22. Composition according to any of the preceding paragraphs    comprising from about 5 wt % to about 40 wt % nonionic surfactants,    and from about 0 wt % to about 5 wt % anionic surfactants.-   23. Composition according to paragraph 22, wherein the nonionic    surfactant is selected from alcohol ethoxylates (AE or AEO), alcohol    propoxylates, propoxylated fatty alcohols (PFA), alkoxylated fatty    acid alkyl esters, such as ethoxylated and/or propoxylated fatty    acid alkyl esters, alkylphenol ethoxylates (APE), nonylphenol    ethoxylates (NPE), alkylpolyglycosides (APG), alkoxylated amines,    fatty acid monoethanolamides (FAM), fatty acid diethanolamides    (FADA), ethoxylated fatty acid monoethanolamides (EFAM),    propoxylated fatty acid monoethanolamides (PFAM), polyhydroxyalkyl    fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine    (glucamides, GA, or fatty acid glucamides, FAGA) and combinations    thereof.-   24. Composition according to any of the preceding composition    paragraphs, wherein the composition further comprises one or more    enzymes selected from the group consisting of proteases, lipases,    cutinases, amylases, carbohydrases, cellulases, pectinases,    mannanases, arabinases, galactanases, xylanases and oxidases.-   25. Composition according to any of the preceding composition    paragraphs, wherein the enzyme is a protease, which is of animal,    vegetable or microbial origin.-   26. Composition according to any of the preceding composition    paragraphs, wherein the protease is chemically modified or protein    engineered.-   27. Composition according to any of the preceding composition    paragraphs, wherein the protease is a serine protease or a    metalloprotease, preferably an alkaline microbial protease or a    trypsin-like protease.-   28. Composition according to any of the preceding composition    paragraphs, wherein the protease is selected from the group    consisting of Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg,    subtilisin 309, subtilisin 147, subtilisin 168, trypsin of bovine    origin, trypsin of porcine origin and Fusarium protease.-   29. Composition according to any of the preceding composition    paragraphs, wherein the composition is capable of reducing adhesion    of EPS from bacteria selected from the group consisting of    Acinetobacter sp., Aeromicrobium sp., Brevundimonas sp.,    Microbacterium sp., Micrococcus luteus, Pseudomonas sp.,    Staphylococcus epidermidis, Staphylococcus aureus and    Stenotrophomonas sp. to a surface, or releasing the bacteria from a    surface to which they adhere.-   30. Composition according to any of the preceding composition    paragraphs, wherein the composition is a bar, a homogenous tablet, a    tablet having two or more layers, a pouch having one or more    compartments, a regular or compact powder, a granule, a paste, a    gel, or a regular, compact or concentrated liquid.-   31. Composition according to any of the preceding composition    paragraphs, wherein the composition is a cleaning composition    selected from liquid detergent, powder detergent and granule    detergent compositions.-   32. A method for laundering an item comprising the steps of:    -   a. Exposing an item to a wash liquor comprising a polypeptide of        paragraphs 46-56 or a composition according to any of paragraphs        11-31;    -   b. Completing at least one wash cycle; and    -   c. Optionally rinsing the item,

wherein the item is a textile.

-   33. A method for deep cleaning of an item, wherein the item is    preferably a textile, said method comprising; exposing an item to a    wash liquor comprising a polypeptide, preferably comprising one or    more of the motif(s) GXDE (SEQ ID NO 18),    [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19),    [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO: 20), WND[SQR][IVL][TLVM]    (SEQ ID NO: 21), QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO: 23) or    NLD[DR]S (SEQ ID NO: 24), wherein the polypeptide is selected from    the group consisting of polypeptides having at least 60% e.g. 80%,    85%, 90%, 95%, 98%, 99% or 100% sequence identity to the polypeptide    shown in SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 and SEQ    ID NO 11.-   34. Method according to paragraph 32 or 33, wherein the pH of the    wash liquor is in the range of 1 to 11.-   35. Method according to any of the preceding method paragraphs,    wherein the pH of the wash liquor is in the range 5.5 to 11, such as    in the range of 7 to 9, in the range of 7 to 8 or in the range of 7    to 8.5.-   36. Method according to any of the preceding method paragraphs,    wherein the temperature of the wash liquor is in the range of 5° C.    to 95° C., or in the range of 10° C. to 80° C., in the range of    10° C. to 70° C., in the range of 10° C. to 60° C., in the range of    10° C. to 50° C., in the range of 15° C. to 40° C., in the range of    20° C. to 40° C., in the range of 15° C. to 30° C. or in the range    of 20° C. to 30° C.-   37. Method according to any of the preceding method paragraphs,    wherein the temperature of the wash liquor is from about 20° C. to    about 40° C.-   38. Method according to any of the preceding method paragraphs,    wherein the temperature of the wash liquor is from about 15° C. to    about 30° C.-   39. Method according to any of the preceding method paragraphs,    wherein stains present on the item is pre-treated with a polypeptide    of paragraphs 46-60 or a detergent composition according to any of    paragraphs 11-31.-   40. Method according to any of the preceding method paragraphs,    wherein stickiness of the item is reduced.-   41. Method according to any of the preceding method paragraphs,    wherein redeposition of soil is reduced.-   42. Method according to any of the preceding method paragraphs,    wherein adherence of soil to the item is reduced or removed.-   43. Method according to any of the preceding method paragraphs,    wherein whiteness of the item is maintained or improved.-   44. Method according to any of the preceding method paragraphs,    wherein malodor is reduced or removed from the item.-   45. Method according to any of the preceding method paragraphs,    wherein the concentration of the polypeptide having hexosaminidase    activity in the wash liquor is at least 0.001 mg of polypeptide,    such as at least 0.05 mg of protein, or at least 1.0 mg of protein,    or at least 1.5 mg of protein per liter of wash liquor, optionally    the concentration of polypeptide in the wash liquor is in the range    0.0002 mg/L to 2 mg/L, such as 0.002 mg/L to 2 mg/L, such as 0.2    mg/L to 2 mg/L or in the range of 0.00001 mg/L to 10 mg/L or in the    range of in the range of 0.0001 mg/L to 10 mg/L, or in the range of    0.001 mg/L to 10 mg/L, or in in the range of 0.01 mg/L to 10 mg/L    per liter of wash liquor, optionally the concentration of the    polypeptide of the invention is 0.00001% to 2 wt %, such as 0.0001    to 0.1 wt %, such as 0.0005 to 0.1 wt %, such as 0.001 to 0.1 wt %,    such as 0.001 to 0.5 wt %, such as 0.002 to 0.5 wt % or 0.0002 to    0.09 wt % in the total detergent concentration.-   46. A polypeptide having hexosaminidase activity, selected from the    group consisting of:    -   a. a polypeptide having at least 60% e.g. 80%, 85%, 90%, 95%,        98%, 99% or 100% sequence identity to the mature polypeptide        shown in SEQ ID NO 7, 8, 9, 10 or 11;    -   b. a polypeptide encoded by a polynucleotide that hybridizes        under low stringency conditions with        -   i. the mature polypeptide coding sequence of SEQ ID NO 1, 3,            5, 12 or 14,        -   ii. the cDNA sequence thereof, or        -   iii. the full-length complement of (i) or (ii);    -   c. a polypeptide encoded by a polynucleotide having at least 60%        e.g. 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to        the mature polypeptide coding sequence of SEQ ID NO 1, 3, 5, 12        or 14 or the cDNA sequence thereof;    -   d. a variant of the mature polypeptide of SEQ ID NO 7, SEQ ID NO        8, SEQ ID NO 9, SEQ ID NO 10 or SEQ ID NO 11 comprising a        substitution, deletion, and/or insertion at one or more        positions; and    -   e. a fragment of the polypeptide of (a), (b), (c), or (d) that        has hexosaminidase activity; and    -   f. a polypeptide comprising one or more of the motif(s) GXDE        (SEQ ID NO 18), [EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ        ID NO 19), [VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO: 20),        WND[SQR][IVL][TLVM] (SEQ ID NO: 21), QSTL (SEQ ID NO 22), NKFFY        (SEQ ID NO: 23), NLD[DR]S (SEQ ID NO: 24).-   47. The polypeptide of paragraph 46, having at least 60%, at least    65%, at least 70%, at least 75%, at least 80%, at least 85%, at    least 90%, at least 91%, at least 92%, at least 93%, at least 94%,    at least 95%, at least 96%, at least 97%, at least 98%, at least 99%    or 100% sequence identity to the mature polypeptide of SEQ ID NO 2,    SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 13 or SEQ ID NO 15, or to the    polypeptide shown in SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID    NO 10 or SEQ ID NO 11.-   48. The polypeptide of paragraph 46 or 47, having at least 60%, at    least 65%, at least 70%, at least 75%, at least 80%, at least 85%,    at least 90%, at least 91%, at least 92%, at least 93%, at least    94%, at least 95%, at least 96%, at least 97%, at least 98%, at    least 99% or 100% sequence identity to the mature polypeptide of SEQ    ID NO 2 or to the mature polypeptide shown in SEQ ID NO 7.-   49. The polypeptide of paragraph 46 or 47, having at least 60%, at    least 65%, at least 70%, at least 75%, at least 80%, at least 85%,    at least 90%, at least 91%, at least 92%, at least 93%, at least    94%, at least 95%, at least 96%, at least 97%, at least 98%, at    least 99% or 100% sequence identity to the mature polypeptide of SEQ    ID NO 4 or to the mature polypeptide shown in SEQ ID NO 8.-   50. The polypeptide of paragraph 46 or 47, having at least 60%, at    least 65%, at least 70%, at least 75%, at least 80%, at least 85%,    at least 90%, at least 91%, at least 92%, at least 93%, at least    94%, at least 95%, at least 96%, at least 97%, at least 98%, at    least 99% or 100% sequence identity to the mature polypeptide of SEQ    ID NO 6 or to the mature polypeptide shown in SEQ ID NO 9.-   51. The polypeptide of paragraph 46 or 47, having at least 60%, at    least 65%, at least 70%, at least 75%, at least 80%, at least 85%,    at least 90%, at least 91%, at least 92%, at least 93%, at least    94%, at least 95%, at least 96%, at least 97%, at least 98%, at    least 99% or 100% sequence identity to the mature polypeptide of SEQ    ID NO 13 or to the mature polypeptide shown in SEQ ID NO 10.-   52. The polypeptide of paragraph 46 or 47, having at least 60%, at    least 65%, at least 70%, at least 75%, at least 80%, at least 85%,    at least 90%, at least 91%, at least 92%, at least 93%, at least    94%, at least 95%, at least 96%, at least 97%, at least 98%, at    least 99% or 100% sequence identity to the mature polypeptide of SEQ    ID NO 15 or to the mature polypeptide shown in SEQ ID NO 11-   53. The polypeptide according to paragraph 46 to 52, which is    encoded by a polynucleotide that hybridizes under low stringency    conditions, low-medium stringency conditions, medium stringency    conditions, medium-high stringency conditions, high stringency    conditions, or very high stringency conditions with    -   i. the mature polypeptide coding sequence of SEQ ID NO 1, 3, 5,        12 or 14,    -   ii. the cDNA sequence thereof, or    -   iii. the full-length complement of (i) or (ii).-   54. The polypeptide according to any of paragraphs 46-53, which is    encoded by a polynucleotide having at least 60%, at least 65%, at    least 70%, at least 75%, at least 80%, at least 85%, at least 90%,    at least 91%, at least 92%, at least 93%, at least 94%, at least    95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%    sequence identity to the mature polypeptide coding sequence of SEQ    ID NO 1, 3, 5, 12, 14 or the cDNA sequence thereof.-   55. The polypeptide according to any of paragraphs 46 to 54,    comprising or consisting of the amino acids sequence shown in SEQ ID    NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 or SEQ ID NO 11 or the    mature polypeptide of SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID    NO 13 or SEQ ID NO 15.-   56. The polypeptide according to any of paragraphs 46 to 55, which    is a variant of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10    or SEQ ID NO 11 comprising a substitution, deletion, and/or    insertion at one or more positions.-   57. A polynucleotide encoding the polypeptide according to any of    paragraphs 46-56.-   58. A nucleic acid construct or expression vector comprising the    polynucleotide of paragraph 57 operably linked to one or more    control sequences that direct the production of the polypeptide in    an expression host.-   59. A recombinant host cell comprising the polynucleotide of    paragraph 57 operably linked to one or more control sequences that    direct the production of the polypeptide.-   60. A method of producing the polypeptide of any of paragraphs    46-56, comprising cultivating a cell, which in its wild-type form    produces the polypeptide, under conditions conducive for production    of the polypeptide.-   61. The method of paragraph 60, further comprising recovering the    polypeptide.-   62. A method of producing a polypeptide according to any of    paragraphs 46-56, comprising cultivating the host cell of paragraph    59 under conditions conducive for production of the polypeptide.-   63. The method of paragraph 62, further comprising recovering the    polypeptide.-   64. A nucleic acid construct or expression vector comprising a gene    encoding a protein operably linked to the polynucleotide of    paragraph 57, wherein the gene is foreign to the polynucleotide    encoding the signal peptide.-   65. A recombinant host cell comprising a gene encoding a protein    operably linked to the polynucleotide of paragraph 57, wherein the    gene is foreign to the polynucleotide encoding the signal peptide.-   66. A method of producing a protein, comprising cultivating a    recombinant host cell comprising a gene encoding a protein operably    linked to the polynucleotide of paragraph 62, wherein the gene is    foreign to the polynucleotide encoding the signal peptide, under    conditions conducive for production of the protein.-   67. The method of paragraph 66, further comprising recovering the    protein.-   68. The recombinant host cell of paragraph 65 further comprising a    polynucleotide encoding a second polypeptide of interest; preferably    an enzyme of interest; more preferably a secreted enzyme of    interest; even more preferably a hydrolase, isomerase, ligase,    lyase, oxidoreductase, or a transferase; and most preferably the    secreted enzyme is an alpha-galactosidase, alpha-glucosidase,    aminopeptidase, amylase, asparaginase, beta-galactosidase,    beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase,    catalase, cellobiohydrolase, cellulase, chitinase, cutinase,    cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase,    esterase, green fluorescent protein, glucano-transferase,    glucoamylase, invertase, laccase, lipase, mannosidase, mutanase,    oxidase, pectinolytic enzyme, peroxidase, phytase,    polyphenoloxidase, proteolytic enzyme, ribonuclease,    transglutaminase, or a xylanase.-   69. The recombinant host cell of paragraph 65, wherein the second    polypeptide of interest is heterologous or homologous to the host    cell.-   70. The recombinant host cell of paragraph 65 or 68, which is a    fungal host cell; preferably a filamentous fungal host cell; more    preferably an Acremonium, Aspergillus, Aureobasidium, Bjerkandera,    Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus,    Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor,    Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,    Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,    Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,    Trametes, or Trichoderma cell; most preferably an Aspergillus    awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus    japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus    oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis    caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta,    Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis    subvermispora, Chrysosporium inops, Chrysosporium keratinophilum,    Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium    pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum,    Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus,    Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,    Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium    heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium    reticulaturn, Fusarium roseum, Fusarium sambucinum, Fusarium    sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum,    Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum,    Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora    thermophila, Neurospora crassa, Penicillium purpurogenum,    Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,    Thielavia terrestris, Trametes villosa, Trametes versicolor,    Trichoderma harzianum, Trichoderma koningii, Trichoderma    longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.-   71. The recombinant host cell of paragraph 65 or 68, which is a    bacterial host cell; preferably a prokaryotic host cell; more    preferably a Gram-positive host cell; even more preferably a    Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,    Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or    Streptomyces host cell; and most preferably a Bacillus alkalophilus,    Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,    Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus    lautus, Bacillus lentus, Bacillus licheniformis, Bacillus    megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus    subtilis, and Bacillus thuringiensis host cell.-   72. Item laundered according to the method of any of paragraphs    32-45.    Some preferred embodiments are summarized in the following    paragraphs:    -   1. A composition comprising at least 0.01 mg of active enzyme        per gram of composition, wherein the polypeptide having        hexosaminidase activity is selected from the group consisting of        a polypeptide having at least 60% sequence identity to the        mature polypeptide of SEQ ID NO: 2, 4 and 6 and at least one        adjunct ingredient.    -   2. The composition of paragraph 1, wherein the polypeptide has        at least 60%, at least 65%, at least 70%, at least 75%, at least        80%, at least 85%, at least 90%, at least 91%, at least 92%, at        least 93%, at least 94%, at least 95%, at least 96%, at least        97%, at least 98%, at least 99% or 100% sequence identity to the        mature polypeptide of SEQ ID NO: 2, 4 and 6.    -   3. The composition of any of paragraphs 1 or 2, comprising or        consisting of SEQ ID NO: 7 or the mature polypeptide of SEQ ID        NO: 2, SEQ ID NO: 8 or the mature polypeptide of SEQ ID NO: 4 or        SEQ ID NO: 9 or the mature polypeptide of SEQ ID NO: 6.    -   4. The composition according to any of paragraphs 1 to 3 wherein        the composition is a cleaning composition such as a laundry or        dish wash composition    -   5. The composition according to paragraph 4, wherein the adjunct        ingredient is selected from,        -   a. at least one builder,        -   b. at least one surfactant, and        -   c. at least one bleach component.    -   6. The composition according to paragraph 5, wherein the        composition comprises at least one builder, wherein the builder        is added in an amount of about 0-65% by weight, preferably about        40-65% by weight, particularly about 20-65% by weight,        particularly from 10% to 50% by weight and wherein the builder        is selected among phosphates, sodium citrate builders, sodium        carbonate, sodium silicate, sodium and zeolites.    -   7. The composition according to paragraph 6 wherein the builder        is selected from citric acid, methyl glycine-N,N-diacetic acid        (MGDA) and/or glutamic-N,N-diacetic acid (GLDA) and mixtures        thereof.    -   8. The composition according to any of the preceding paragraphs        comprising 1-40 wt %, preferably from 0.5-30 wt %, of at least        one bleaching component, wherein the bleach component includes a        percarbonate and bleach catalyst, preferably a manganese        compound.    -   9. The composition according to paragraph 8 wherein at least one        bleach component is a peroxide, preferably percabonate and a        catalyst preferably a metal-containing bleach catalyst such as        1,4,7-trimethyl-1,4,7-triazacyclononane or manganese (II)        acetate tetrahydrate (MnTACN).    -   10. The composition according to any of the previous paragraphs,        wherein the composition comprise at least one surfactant wherein        the surfactant is anionic and/or nonionic.    -   11. The composition according to paragraph 10, wherein the        composition comprises from about 5 wt % to about 50 wt %, from        about 5 wt % to about 40 wt %, from about 5 wt % to about 30 wt        %, from about 5 wt % to about 20 wt %, from about 5 wt % to        about 10 wt % anionic surfactant.    -   12. The composition according to any of paragraphs 10 or 11,        wherein the composition comprises from about 5 wt % to about 50        wt %, from about 5 wt % to about 40 wt %, from about 5 wt % to        about 30 wt %, from about 5 wt % to about 20 wt %, from about 5        wt % to about 10 wt % nonionic surfactant.    -   13. The composition according to any of paragraphs 10 to 12,        wherein the anionic surfactant is selected from linear        alkylbenzenesulfonates (LAS) isomers of LAS, alcohol ether        sulfate (AEO, AEOS) and sodium lauryl ether sulfate and sodium        laureth sulfate (SLES).    -   14. The composition according to any of paragraphs 10 to 13,        wherein the nonionic surfactant is selected from alcohol        ethoxylates (AE or AEO), alcohol propoxylates, alcohol        propoxylates, propoxylated fatty alcohols (PFA), alkoxylated        fatty acid alkyl esters, such as ethoxylated and/or propoxylated        fatty acid alkyl esters, alkylphenol ethoxylates (APE),        nonylphenol ethoxylates (NPE), alkylpolyglycosides (APG),        alkoxylated amines, fatty acid monoethanolamides (FAM), fatty        acid diethanolamides (FADA), ethoxylated fatty acid        monoethanolamides (EFAM), propoxylated fatty acid        monoethanolamides (PFAM), polyhydroxyalkyl fatty acid amides,        N-acyl N-alkyl derivatives of glucosamine (glucamides, GA, or        fatty acid glucamides, FAGA) and combinations thereof.    -   15. Use of a composition of any of paragraphs 1 to 14 for        deep-cleaning of an item, wherein the item is a textile.    -   16. A laundering method for laundering an item comprising the        steps of:        -   Exposing an item to a wash liquor comprising a polypeptide            selected from the group consisting of a polypeptide having            at least 60% sequence identity to the mature polypeptide of            SEQ ID NO: 2, 4 and 6 or a detergent composition according            to any of paragraphs 1 to 14;        -   Completing at least one wash cycle; and        -   Optionally rinsing the item,

wherein the item is a textile.

-   -   17. Use of a polypeptide of the Terribacillus clade, wherein the        polypeptide has hexosaminidase activity in a cleaning process,        such as laundry and/or dish wash.    -   18. Use of a polypeptide of the Terribacillus clade, wherein the        polypeptide has hexosaminidase activity for deep cleaning of an        item, wherein the item is a textile.    -   19. Use according to paragraph 17 for preventing, reducing or        removing stickiness of the item.    -   20. Use according to any of paragraphs 17 or 18 for preventing,        reducing or removing redeposition of soil during a wash cycle.    -   21. Use according to any of the preceding paragraphs, wherein        the polypeptide is selected from the group consisting of a        polypeptide having at least 60% sequence identity to the mature        polypeptide of SEQ ID NO: 2, 4 and 6 and at least one adjunct        ingredient.    -   22. The use of paragraph 21, wherein the polypeptide has at        least 60%, at least 65%, at least 70%, at least 75%, at least        80%, at least 85%, at least 90%, at least 91%, at least 92%, at        least 93%, at least 94%, at least 95%, at least 96%, at least        97%, at least 98%, at least 99% or 100% sequence identity to the        mature polypeptide of SEQ ID NO: 2, 4 and 6.

It should be understood that every maximum numerical limitation giventhroughout this specification includes every lower numerical limitation,as if such lower numerical limitations were expressly written herein.Every minimum numerical limitation given throughout this specificationwill include every higher numerical limitation, as if such highernumerical limitations were expressly written herein. Every numericalrange given throughout this specification will include every narrowernumerical range that falls within such broader numerical range, as ifsuch narrower numerical ranges were all expressly written herein.

Assays and Detergent Compositions

Detergent Compositions

The below mentioned detergent composition may be used in combinationwith the enzyme of the invention.

Biotex Black (Liquid)

5-15% Anionic surfactants, <5% Nonionic surfactants, perfume, enzymes,DMDM and hydantoin.

Composition of Ariel Sensitive White & Color, Liquid DetergentComposition

Aqua, Alcohol Ethoxy Sulfate, Alcohol Ethoxylate, Amino Oxide, CitricAcid, C12-18 topped palm kernel fatty acid, Protease, Glycosidase,Amylase, Ethanol, 1,2 Propanediol, Sodium Formate, Calcium Chloride,Sodium hydroxide, Silicone Emulsion, Trans-sulphated EHDQ (theingredients are listed in descending order).

Composition of WFK IEC-A Model Detergent (Powder)

Ingredients: Linear sodium alkyl benzene sulfonate 8.8%, Ethoxylatedfatty alcohol C12-18 (7 EO) 4.7%, Sodium soap 3.2%, Anti foam DC2-424853.9%, Sodium aluminium silicate zeolite 4A 28.3%, Sodium carbonate11.6%, Sodium salt of a copolymer from acrylic and maleic acid (SokalanCP5) 2.4%, Sodium silicate 3.0%, Carboxymethylcellulose 1.2%, Dequest2066 2.8%, Optical whitener 0.2%, Sodium sulfate 6.5%, Protease 0.4%.

Composition of Model Detergent A (Liquid)

Ingredients: 12% LAS, 11% AEO Biosoft N25-7 (NI), 5% AEOS (SLES), 6% MPG(monopropylene glycol), 3% ethanol, 3% TEA, 2.75% coco soap, 2.75% soyasoap, 2% glycerol, 2% sodium hydroxide, 2% sodium citrate, 1% sodiumformate, 0.2% DTMPA and 0.2% PCA (all percentages are w/w)

Composition of Model Detergent N (Liquid)

Ingredients: NaOH 0.87%, MPG (Monopropylenglycol) 6%, Glycerol 2%,Soap-soy 2.75%, Soap-coco 2.75%, PCA (Sokalon CP-5) 0.2%, AEO BiosoftN25-7(NI) 16%, Sodium formiate 1%, Sodium Citrate 2%, DTMPA 0.2%,Ethanol (96%) 3%, adjustment of pH with NaOH or Citric acid as water to100% (all percentages are w/w (weight volume).

Composition of Persil Universal Gel

Ingredients: 15-30% Anionic surfactants, 5-15% Nonionic surfactant, <5%Phosphonate, soap, Amyl cinnamal, Butylphenyl methylpropional, limonene,Gernaiol, Optical brightener, enzymes.

Composition of Ariel Actilift (Liquid)

Ingredients: 5-15% Anionic surfactants; <5% Non-ionic surfactants,Phosphonates, Soap; Enzymes, Optical brighteners, Benzisothiazolinone,Methylisothiazolinone, Perfumes, Alpha-isomethyl ionone, Citronellol,Geraniol, Linalool.

Composition of Ariel Actilift Colour&Style (Liquid)

Ingredients: 5-15% Anionic surfactants; <5% Non-ionic surfactants,Phosphonates, Soap; Enzymes, Perfumes, Benzisothiazolinone,Methylisothiazolinone, Alpha-isomethyl ionone, Butylphenylmethylpropional, Citronellol, Geraniol, Linalool.

Composition of Persil Small & Mighty (Liquid)

Ingredients: 15-30% Anionic surfactants, Non-ionic surfacts, 5-15% Soap,<5% Polycarboxylates, Perfume, Phosphates, Optical Brighteners

Persil 2 in 1 with Comfort Passion Flower Powder

Sodium sulfate, Sodium carbonate, Sodium Dodecylbenzenesulfonate,Bentonite, Sodium Carbonate Peroxide, Sodium Silicate, Zeolite, Aqua,Citric acid, TAED, C12-15 Pareth-7, Stearic Acid, Parfum, Sodium AcrylicAcid/MA Copolymer, Cellulose Gum, Corn Starch Modified, Sodium chloride,Tetrasodium Etidronate, Calcium Sodium EDTMP, DisodiumAnilinomorpholinotriazinyl-aminostilbenesulfonate, Sodium bicarbonate,Phenylpropyl Ethyl Methicone, Butylphenyl Methylpropional, GlycerylStearates, Calcium carbonate, Sodium Polyacrylate, Alpha-IsomethylIonone, Disodium Distyrylbiphenyl Disulfonate, Cellulose, Protease,Limonene, PEG-75, Titanium dioxide, Dextrin, Sucrose, Sodium PolyarylSulphonate, CI 12490, CI 45100, CI 42090, Sodium Thiosulfate, CI 61585.

Persil Biological Powder

Sucrose, Sorbitol, Aluminum Silicate, Polyoxymethylene Melamine, SodiumPolyaryl Sulphonate, CI 61585, CI 45100, Lipase, Amylase, Xanthan gum,Hydroxypropyl methyl cellulose, CI 12490, Disodium DistyrylbiphenylDisulfonate, Sodium Thiosulfate, CI 42090, Mannanase, CI 11680,Etidronic Acid, Tetrasodium EDTA.

Persil Biological Tablets

Sodium carbonate, Sodium Carbonate Peroxide, Sodium bicarbonate,Zeolite, Aqua, Sodium Silicate, Sodium Lauryl Sulfate, Cellulose, TAED,Sodium Dodecylbenzenesulfonate, Hemicellulose, Lignin, Lauryl Glucoside,Sodium Acrylic Acid/MA Copolymer, Bentonite, Sodium chloride, Parfum,Tetrasodium Etidronate, Sodium sulfate, Sodium Polyacrylate,Dimethicone, Disodium Anilinomorpholinotriazinylaminostilbenesulfonate,Dodecylbenzene Sulfonic Acid, Trimethylsiloxysilicate, Calciumcarbonate, Cellulose, PEG-75, Titanium dioxide, Dextrin, Protease, CornStarch Modified, Sucrose, CI 12490, Sodium Polyaryl Sulphonate, SodiumThiosulfate, Amylase, Kaolin,

Persil Colour Care Biological Powder

Subtilisin, Imidazolidinone, Hexyl Cinnamal, Sucrose, Sorbitol, AluminumSilicate, Polyoxymethylene Melamine, CI 61585, CI 45100, Lipase,Amylase, Xanthan gum, Hydroxypropyl methyl cellulose, CI 12490, DisodiumDistyrylbiphenyl Disulfonate, Sodium Thiosulfate, CI 42090, Mannanase,CI 11680, Etidronic Acid, Tetrasodium EDTA.

Persil Colour Care Biological Tablets

Sodium bicarbonate, Sodium carbonate, Zeolite, Aqua, Sodium Silicate,Sodium Lauryl Sulfate, Cellulose Gum, Sodium Dodecylbenzenesulfonate,Lauryl Glucoside, Sodium chloride, Sodium Acrylic Acid/MA Copolymer,Parfum, Sodium Thioglycolate, PVP, Sodium sulfate, TetrasodiumEtidronate, Sodium Polyacrylate, Dimethicone, Bentonite, DodecylbenzeneSulfonic Acid, Trimethylsiloxysilicate, Calcium carbonate, Cellulose,PEG-75, Titanium dioxide, Dextrin, Protease, Corn Starch Modified,Sucrose, Sodium Thiosulfate, Amylase, CI 74160, Kaolin.

Persil Dual Action Capsules Bio

MEA-Dodecylbenzenesulfonate, MEA-Hydrogenated Cocoate, 012-15 Pareth-7,Dipropylene Glycol, Aqua, Tetrasodium Etidronate, Polyvinyl Alcohol,Glycerin, Aziridine, homopolymer ethoxylated, Propylene glycol, Parfum,Sodium Diethylenetriamine Pentamethylene Phosphonate, Sorbitol,MEA-Sulfate, Ethanolamine, Subtilisin, Glycol, ButylphenylMethylpropional, Boronic acid, (4-formylphenyl), Hexyl Cinnamal,Limonene, Linalool, Disodium Distyrylbiphenyl Disulfonate,Alpha-Isomethyl Ionone, Geraniol, Amylase, Polymeric Blue Colourant,Polymeric Yellow Colourant, Talc, Sodium chloride, Benzisothiazolinone,Mannanase, Denatonium Benzoate.

Persil 2 in 1 with Comfort Sunshiny Days Powder

Sodium sulfate, Sodium carbonate, Sodium Dodecylbenzenesulfonate,Bentonite, Sodium Carbonate Peroxide, Sodium Silicate, Zeolite, Aqua,Citric acid, TAED, C12-15 Pareth-7, Parfum, Stearic Acid, Sodium AcrylicAcid/MA Copolymer, Cellulose Gum, Corn Starch Modified, Sodium chloride,Tetrasodium Etidronate, Calcium Sodium EDTMP, DisodiumAnilinomorpholinotriazinyl-aminostilbenesulfonate, Sodium bicarbonate,Phenylpropyl Ethyl Methicone, Butylphenyl Methylpropional, GlycerylStearates, Calcium carbonate, Sodium Polyacrylate, Geraniol, DisodiumDistyrylbiphenyl Disulfonate, Cellulose, Protease, PEG-75, Titaniumdioxide, Dextrin, Sucrose, Sodium Polyaryl Sulphonate, CI 12490, CI45100, CI 42090, Sodium Thiosulfate, CI 61585.

Persil Small & Mighty 2in1 with Comfort Sunshiny Days

Aqua, C12-15 Pareth-7, Sodium Dodecylbenzenesulfonate, Propylene glycol,Sodium Hydrogenated Cocoate, Triethanolamine, Glycerin, TEA-HydrogenatedCocoate, Parfum, Sodium chloride, Polyquaternium-10, PVP, Polymeric PinkColourant, Sodium sulfate, Disodium Distyrylbiphenyl Disulfonate,Butylphenyl Methylpropional, Styrene/Acrylates Copolymer, HexylCinnamal, Citronellol, Eugenol, Polyvinyl Alcohol, Sodium acetate,Isopropyl alcohol, Polymeric Yellow Colourant, Sodium Lauryl Sulfate.

Persil Small & Mighty Bio

Aqua, MEA-Dodecylbenzenesulfonate, Propylene glycol, Sodium LaurethSulfate, C12-15 Pareth-7, TEA-Hydrogenated Cocoate, MEA-Citrate,Aziridine homopolymer ethoxylated, MEA-Etidronate, Triethanolamine,Parfum, Acrylates Copolymer, Sorbitol, MEA-Sulfate, Sodium Sulfite,Disodium Distyrylbiphenyl Disulfonate, Butylphenyl Methylpropional,Styrene/Acrylates Copolymer, Citronellol, Sodium sulfate, Peptides,salts, sugars from fermentation (process), Subtilisin, Glycerin, Boronicacid, (4-formylphenyl), Geraniol, Pectate Lyase, Amylase, Sodium LaurylSulfate, Mannanase, CI 42051.

Persil Small & Mighty Capsules Biological

MEA-Dodecylbenzenesulfonate, MEA-Hydrogenated Cocoate, C12-15 Pareth-7,Dipropylene Glycol, Aqua, Glycerin, Polyvinyl Alcohol, Parfum, Aziridinehomopolymer ethoxylated, Sodium Diethylenetriamine PentamethylenePhosphonate, Propylene glycol, Sorbitol, MEA-Sulfate, Ethanolamine,Subtilisin, Glycol, Butylphenyl Methylpropional, Hexyl Cinnamal, Starch,Boronic acid, (4-formylphenyl), Limonene, Linalool, DisodiumDistyrylbiphenyl Disulfonate, Alpha-Isomethyl Ionone, Geraniol, Amylase,Talc, Polymeric Blue Colourant, Sodium chloride, Benzisothiazolinone,Denatonium Benzoate, Polymeric Yellow Colourant, Mannanase.

Persil Small & Mighty Capsules Colour Care

MEA-Dodecylbenzenesulfonate, MEA-Hydrogenated Cocoate, C12-15 Pareth-7,Dipropylene Glycol, Aqua, Glycerin, Polyvinyl Alcohol, Parfum, Aziridinehomopolymer ethoxylated, Sodium Diethylenetriamine PentamethylenePhosphonate, Propylene glycol, MEA-Sulfate, Ethanolamine, PVP, Sorbitol,Butylphenyl Methylpropional, Subtilisin, Hexyl Cinnamal, Starch,Limonene, Linalool, Boronic acid, (4-formylphenyl), Alpha-IsomethylIonone, Geraniol, Talc, Polymeric Blue Colourant, Denatonium Benzoate,Polymeric Yellow Colourant.

Persil Small & Mighty Colour Care Aqua, MEA-Dodecylbenzenesulfonate,Propylene glycol, Sodium Laureth Sulfate, C12-15 Pareth-7,TEA-Hydrogenated Cocoate, MEA-Citrate, Aziridine homopolymerethoxylated, MEA-Etidronate, Triethanolamine, Parfum, AcrylatesCopolymer, Sorbitol, MEA-Sulfate, Sodium Sulfite, Glycerin, ButylphenylMethylpropional, Citronellol, Sodium sulfate, Peptides, salts, sugarsfrom fermentation (process), Styrene/Acrylates Copolymer, Subtilisin,Boronic acid, (4-formylphenyl), Geraniol, Pectate Lyase, Amylase, SodiumLauryl Sulfate, Mannanase, CI 61585, CI 45100.Composition of Fairy Non Bio (Liquid)

Ingredients: 15-30% Anionic Surfactants, 5-15% Non-Ionic Surfactants,Soap, Benzisothiazolinone, Methylisothiazolinone, Perfumes

Composition of Model Detergent T (Powder)

Ingredients: 11% LAS, 2% AS/AEOS, 2% soap, 3% AEO, 15.15% sodiumcarbonate, 3% sodium slilcate, 18.75% zeolite, 0.15% chelant, 2% sodiumcitrate, 1.65% AA/MA copolymer, 2.5% CMC and 0.5% SRP (all percentagesare w/w).

Composition of Model Detergent X (Powder)

Ingredients: 16.5% LAS, 15% zeolite, 12% sodium disilicate, 20% sodiumcarbonate, 1% sokalan, 35.5% sodium sulfate (all percentages are w/w).

Composition of Ariel Actilift Colour&Style (Powder)

Ingredients: 15-30% Anionic surfactants, <5% Non-ionic surfactants,Phosphonates, Polycarboxylates, Zeolites; Enzymes, Perfumes, Hexylcinnamal.

Composition of Ariel Actilift (Powder)

Ingredients: 5-15% Anionic surfactants, Oxygen-based bleaching agents,<5% Non-ionic surfactants, Phosphonates, Polycarboxylates, Zeolites,Optical brighteners, Enzymes, Perfumes, Butylphenyl Methylpropional,Coumarin, Hexyl Cinnamal

Composition of Persil Megaperls (Powder)

Ingredients: 15-30% of the following: anionic surfactants, oxygen-basedbleaching agent and zeolites, less than 5% of the following: non-ionicsurfactants, phosphonates, polycarboxylates, soap, Further ingredients:Perfumes, Hexyl cinnamal, Benzyl salicylate, Linalool, opticalbrighteners, Enzymes and Citronellol.

Gain Liquid, Original:

Ingredients: Water, Alcohol Ethoxysulfate, Diethylene Glycol, AlcoholEthoxylate, Ethanolamine, Linear Alkyl Benzene Sulfonate, Sodium FattyAcids, Polyethyleneimine Ethoxylate, Citric Acid, Borax, Sodium CumeneSulfonate, Propylene Glycol, DTPA, Disodium Diaminostilbene Disulfonate,Dipropylethyl Tetramine, Sodium Hydroxide, Sodium Formate, CalciumFormate, Dimethicone, Amylase, Protease, Liquitint™, Hydrogenated CastorOil, Fragrance.

Tide Liquid, Original:

Ingredients: Linear alkylbenzene sulfonate, propylene glycol, citricacid, sodium hydroxide, borax, ethanolamine, ethanol, alcohol sulfate,polyethyleneimine ethoxylate, sodium fatty acids, diquaterniumethoxysulfate, protease, diethylene glycol, laureth-9,alkyldimethylamine oxide, fragrance, amylase, disodium diaminostilbenedisulfonate, DTPA, sodium formate, calcium formate, polyethylene glycol4000, mannanase, Liquitint™ Blue, dimethicone.

Liquid Tide, Free and Gentle:

Water, sodium alcoholethoxy sulfate, propylene glycol, borax, ethanol,linear alkylbenzene sulfonate sodium, salt, polyethyleneimineethoxylate, diethylene glycol, trans sulphated & ethoxylatedhexamethylene diamine, alcohol ethoxylate, linear alkylbenzenesulfonate, MEA salt, sodium formate, sodium alkyl sulfate, DTPA, amineoxide, calcium formate, disodium diaminostilbene, disulfonate, amylase,protease, dimethicone, benzisothiazolinone

Tide Coldwater Liquid, Fresh Scent:

Water, alcoholethoxy sulfate, linear alkylbenzene sulfonate, diethyleneglycol, propylene glycol, ethanolamine, citric acid, Borax, alcoholsulfate, sodium hydroxide, polyethyleneimine, ethoxylate, sodium fattyacids, ethanol, protease, Laureth-9, diquaternium ethoxysulfate,lauramine oxide, sodium cumene, sulfonate, fragrance, DTPA, amylase,disodium, diaminostilbene, disulfonate, sodium formate, disodiumdistyrylbiphenyl disulfonate, calcium formate, polyethylene glycol 4000,mannanase, pectinase, Liquitint™ Blue, dimethicone

Tide TOTALCARE™ Liquid, Cool Cotton:

Water, alcoholethoxy sulfate, propylene glycol, sodium fatty acids,laurtrimonium chloride, ethanol, sodium hydroxide, sodium cumenesulfonate, citric acid, ethanolamine, diethylene glycol, siliconepolyether, borax, fragrance, polyethyleneimine ethoxylate, protease,Laureth-9, DTPA, polyacrylamide quaternium chloride, disodiumdiaminostilbene disulfonate, sodium formate, Liquitint™ Orange,dipropylethyl tetraamine, dimethicone, cellulase, Liquid Tide PlusBleach Alternative™, Vivid White and Bright, Original and Clean BreezeWater, sodium alcoholethoxy sulfate, sodium alkyl sulfate, MEA citrate,linear alkylbenzene sulfonate, MEA salt, propylene glycol, diethyleneglycol, polyethyleneimine ethoxylate, ethanol, sodium fatty acids,ethanolamine, lauramine oxide, borax, Laureth-9, DTPA, sodium cumenesulfonate, sodium formate, calcium formate, linear alkylbenzenesulfonate, sodium salt, alcohol sulfate, sodium hydroxide, diquaterniumethoxysulfate, fragrance, amylase, protease, mannanase, pectinase,disodium diaminostilbene disulfonate, benzisothiazolinone, Liquitint™Blue, dimethicone, dipropylethyl tetraamine.

Liquid Tide HE, Original Scent:

Water, Sodium alcoholethoxy sulfate, MEA citrate, Sodium Alkyl Sulfate,alcohol ethoxylate, linear alkylbenzene sulfonate, MEA salt, sodiumfatty acids, polyethyleneimine ethoxylate, diethylene glycol, propyleneglycol, diquaternium ethoxysulfate, borax, polyethyleneimine, ethoxylatepropoxylate, ethanol, sodium cumene sulfonate, fragrance, DTPA, disodiumdiaminostilbene disulfonate, Mannanase, cellulase, amylase, sodiumformate, calcium formate, Lauramine oxide, Liquitint™ Blue,Dimethicone/polydimethyl silicone.

Tide TOTALCARE HE Liquid, Renewing Rain:

Water, alcoholethoxy sulfate, linear alkylbenzene sulfonate, alcoholethoxylate, citric acid, Ethanolamine, sodium fatty acids, diethyleneglycol, propylene glycol, sodium hydroxide, borax, polyethyleneimineethoxylate, silicone polyether, ethanol, protease, sodium cumenesulfonate, diquaternium ethoxysulfate, Laureth-9, fragrance, amylase,DTPA, disodium diaminostilbene disulfonate, disodium distyrylbiphenyldisulfonate, sodium formate, calcium formate, mannanase, Liquitint™Orange, dimethicone, polyacrylamide quaternium chloride, cellulase,dipropylethyl tetraamine.

Tide Liquid HE Free:

Water, alcoholethoxy sulfate, diethylene glycol, monoethanolaminecitrate, sodium formate, propylene glycol, linear alkylbenzenesulfonates, ethanolamine, ethanol, polyethyleneimine ethoxylate,amylase, benzisothiazolin, borax, calcium formate, citric acid,diethylenetriamine pentaacetate sodium, dimethicone, diquaterniumethoxysulfate, disodium diaminostilbene disulfonate, Laureth-9,mannanase, protease, sodium cumene sulfonate, sodium fatty acids.

Tide Coldwater HE Liquid, Fresh Scent:

Water, alcoholethoxy sulfate, MEA Citrate, alcohol sulfate, Alcoholethoxylate, Linear alkylbenzene sulfonate MEA, sodium fatty acids,polyethyleneimine ethoxylate, diethylene glycol, propylene glycol,diquaternium ethoxysulfate, borax, polyethyleneimine ethoxylatepropoxylate, ethanol, sodium cumene sulfonate, fragrance, DTPA, disodiumdiaminostilbene disulfonate, protease, mannanase, cellulase, amylase,sodium formate, calcium formate, lauramine oxide, Liquitint™ Blue,dimethicone.

Tide for Coldwater HE Free Liquid:

Water, sodium alcoholethoxy sulfate, MEA Citrate, Linear alkylbenzenesulfonate: sodium salt, Alcohol ethoxylate, Linear alkylbenzenesulfonate: MEA salt, sodium fatty acids, polyethyleneimine ethoxylate,diethylene glycol, propylene glycol, diquaternium ethoxysulfate, Borax,protease, polyethyleneimine ethoxylate propoxylate, ethanol, sodiumcumene sulfonate, Amylase, citric acid, DTPA, disodium diaminostilbenedisulfonate, sodium formate, calcium formate, dimethicone.

Tide Simply Clean & Fresh:

Water, alcohol ethoxylate sulfate, linear alkylbenzene sulfonateSodium/Mea salts, propylene glycol, diethylene glycol, sodium formate,ethanol, borax, sodium fatty acids, fragrance, lauramine oxide, DTPA,Polyethylene amine ethoxylate, calcium formate, disodium diaminostilbenedisulfonate, dimethicone, tetramine, Liquitint™ Blue.

Tide Pods, Ocean Mist, Mystic Forest, Spring Meadow:

Linear alkylbenzene sulfonates, C12-16 Pareth-9, propylene glycol,alcoholethoxy sulfate, water, polyethyleneimine ethoxylate, glycerine,fatty acid salts, PEG-136 polyvinyl acetate, ethylene Diamine disuccinicsalt, monoethanolamine citrate, sodium bisulfite, diethylenetriaminepentaacetate sodium, disodium distyrylbiphenyl disulfonate, calciumformate, mannanase, exyloglucanase, sodium formate, hydrogenated castoroil, natalase, dyes, termamyl, subtilisin, benzisothiazolin, perfume.

Tide to Go:

Deionized water, Dipropylene Glycol Butyl Ether, Sodium Alkyl Sulfate,Hydrogen Peroxide, Ethanol, Magnesium Sulfate, Alkyl Dimethyl AmineOxide, Citric Acid, Sodium Hydroxide, Trimethoxy Benzoic Acid,Fragrance.

Tide Stain Release Liquid:

Water, Alkyl Ethoxylate, Linear Alkylbenzenesulfonate, HydrogenPeroxide, Diquaternium Ethoxysulfate, Ethanolamine, DisodiumDistyrylbiphenyl Disulfonate, tetrabutyl Ethylidinebisphenol, F&DCYellow 3, Fragrance.

Tide Stain Release Powder:

Sodium percarbonate, sodium sulfate, sodium carbonate, sodiumaluminosilicate, nonanoyloxy benzene sulfonate, sodium polyacrylate,water, sodium alkylbenzenesulfonate, DTPA, polyethylene glycol, sodiumpalmitate, amylase, protease, modified starch, FD&C Blue 1, fragrance.

Tide Stain Release, Pre-Treater Spray:

Water, Alkyl Ethoxylate, MEA Borate, Linear Alkylbenzenesulfonate,Propylene Glycol, Diquaternium Ethoxysulfate, Calcium Chlorideenzyme,Protease, Ethanolamine, Benzoisothiazolinone, Amylase, Sodium Citrate,Sodium Hydroxide, Fragrance.

Tide to Go Stain Eraser:

Water, Alkyl Amine Oxide, Dipropylene Glycol Phenyl Ether, HydrogenPeroxide, Citric Acid, Ethylene Diamine Disuccinic Acid Sodium salt,Sodium Alkyl Sulfate, Fragrance.

Tide Boost with Oxi:

Sodium bicarbonate, sodium carbonate, sodium percarbonate, alcoholethoxylate, sodium chloride, maleic/acrylic copolymer, nonanoyloxybenzene sulfonate, sodium sulfate, colorant, diethylenetriaminepentaacetate sodium salt, hydrated aluminosilicate (zeolite),polyethylene glycol, sodium alkylbenzene sulfonate, sodium palmitate,starch, water, fragrance.

Tide Stain Release Boost Duo Pac:

Polyvinyl Alcoholpouch film, wherein there is packed a liquid part and apowder part: Liquid Ingredients: Dipropylene Glycol, diquaterniumEthoxysulfate, Water, Glycerin, Liquitint™ Orange, Powder Ingredients:sodium percarbonate, nonanoyloxy benzene sulfonate, sodium carbonate,sodium sulfate, sodium aluminosilicate, sodium polyacrylate, sodiumalkylbenzenesulfonate, maleic/acrylic copolymer, water, amylase,polyethylene glycol, sodium palmitate, modified starch, protease,glycerine, DTPA, fragrance.

Tide Ultra Stain Release:

Water, sodium alcoholethoxy sulfate, linear alkyl benzene sulfonate,sodium/MEA salts, MEA citrate, propylene glycol, polyethyleneimineethoxylate, ethanol, diethylene glycol, polyethyleneiminepropoxyethoxylate, sodium fatty acids, protease, borax, sodium cumenesulfonate, DTPA, fragrance, amylase, disodium diaminostilbenedisulfonate, calcium formate, sodium formate, gluconase, dimethicone,Liquitint™ Blue, mannanase.

Ultra Tide with a Touch of Downy® Powdered Detergent, April Fresh/CleanBreeze/April Essence:

Sodium Carbonate, Sodium Aluminosilicate, Sodium Sulfate, LinearAlkylbenzene

Sulfonate, Bentonite, Water, Sodium Percarbonate, Sodium Polyacrylate,Silicate, Alkyl Sulfate, Nonanoyloxybenzenesulfonate, DTPA, PolyethyleneGlycol 4000, Silicone, Ethoxylate, fragrance, Polyethylene Oxide,Palmitic Acid, Disodium Diaminostilbene Disulfonate, Protease,Liquitint™ Red, FD&C Blue 1, Cellulase.

Ultra Tide with a Touch of Downy Clean Breeze:

Water, sodium alcoholethoxy sulfate, MEA citrate, linear alkyl benzenesulfonate: sodium/MEA salts, propylene glycol, polyethyleneimineethoxylate, ethanol, diethylene glycol, polyethyleneimine,propoxyethoxylate, diquaternium ethoxysulfate, alcohol sulfate,dimethicone, fragrance, borax, sodium fatty acids, DTPA, protease,sodium bisulfite, disodium diaminostilbene disulfonate, amylase,gluconase, castor oil, calcium formate, MEA, styrene acrylate copolymer,sodium formate, Liquitint™ Blue.

Ultra Tide with Downy Sun Blossom:

Water, sodium alcoholethoxy sulfate, MEA citrate, linear alkyl benzenesulfonate: sodium/MEA salts, propylene glycol, ethanol, diethyleneglycol, polyethyleneimine propoxyethoxylate, polyethyleneimineethoxylate, alcohol sulfate, dimethicone, fragrance, borax, sodium fattyacids, DTPA, protease, sodium bisulfite, disodium diaminostilbenedisulfonate, amylase, castor oil, calcium formate, MEA, styrene acrylatecopolymer, propanaminium propanamide, gluconase, sodium formate,Liquitint™ Blue.

Ultra Tide with Downy April Fresh/Sweet Dreams:

Water, sodium alcoholethoxy sulfate, MEA citrate, linear alkyl benzenesulfonate: sodium/MEA salts, propylene glycol, polyethyleneimineethoxylate, ethanol, diethylene glycol, polyethyleneiminpropoxyethoxylate, diquaternium ethoxysulfate, alcohol sulfate,dimethicone, fragrance, borax, sodium fatty acids, DTPA, protease,sodium bisulfite, disodium diaminostilbene disulfonate, amylase,gluconase, castor oil, calcium formate, MEA, styrene acrylate copolymer,propanaminium propanamide, sodium formate, Liquitint™ Blue.

Ultra Tide Free Powdered Detergent:

Sodium Carbonate, Sodium Aluminosilicate, Alkyl Sulfate, Sodium Sulfate,Linear Alkylbenzene Sulfonate, Water, Sodium polyacrylate, Silicate,Ethoxylate, Sodium percarbonate, Polyethylene Glycol 4000, Protease,Disodium Diaminostilbene Disulfonate, Silicone, Cellulase.

Ultra Tide Powdered Detergent, Clean Breeze/Spring Lavender/MountainSpring:

Sodium Carbonate, Sodium Aluminosilicate, Sodium Sulfate, LinearAlkylbenzene Sulfonate, Alkyl Sulfate, Sodium Percarbonate, Water,Sodium Polyacrylate, Silicate, Nonanoyloxybenzenesulfonate, Ethoxylate,Polyethylene Glycol 4000, Fragrance, DTPA, Disodium DiaminostilbeneDisulfonate, Palmitic Acid, Protease, Silicone, Cellulase.

Ultra Tide HE (High Efficiency) Powdered Detergent, Clean Breeze:

Sodium Carbonate, Sodium Aluminosilicate, Sodium Sulfate, LinearAlkylbenzene Sulfonate, Water, Nonanoyloxybenzenesulfonate, AlkylSulfate, Sodium Polyacrylate, Silicate, Sodium Percarbonate, Ethoxylate,Polyethylene Glycol 4000, Fragrance, DTPA, Palmitic Acid, DisodiumDiaminostilbene Disulfonate, Protease, Silicone, Cellulase.

Ultra Tide Coldwater Powdered Detergent, Fresh Scent:

Sodium Carbonate, Sodium Aluminosilicate, Sodium Sulfate, SodiumPercarbonate, Alkyl Sulfate, Linear Alkylbenzene Sulfonate, Water,Nonanoyloxybenzenesulfonate, Sodium Polyacrylate, Silicate, Ethoxylate,Polyethylene Glycol 4000, DTPA, Fragrance, Natalase, Palmitic Acid,Protease, Disodium, Diaminostilbene Disulfonate, FD&C Blue 1, Silicone,Cellulase, Alkyl Ether Sulfate.

Ultra Tide with Bleach Powdered Detergent, Clean Breeze:

Sodium Carbonate, Sodium Aluminosilicate, Sodium Sulfate, LinearAlkylbenzene Sulfonate, Sodium Percarbonate,Nonanoyloxybenzenesulfonate, Alkyl Sulfate, Water, Silicate, SodiumPolyacrylate, Ethoxylate, Polyethylene Glycol 4000, Fragrance, DTPA,Palmitic Acid, Protease, Disodium Diaminostilbene Disulfonate, Silicone,FD&C Blue 1, Cellulase, Alkyl Ether Sulfate.

Ultra Tide with Febreeze Freshness™ Powdered Detergent, Spring Renewal:

Sodium Carbonate, Sodium Aluminosilicate, Sodium Sulfate, LinearAlkylbenzene Sulfonate, Sodium Percarbonate, Alkyl Sulfate, Water,Sodium Polyacrylate, Silicate, Nonanoyloxybenzenesulfonate, Ethoxylate,Polyethylene Glycol 4000, DTPA, Fragrance, Cellulase, Protease, DisodiumDiaminostilbene Disulfonate, Silicone, FD&C Blue 1.

Liquid Tide Plus with Febreeze Freshness—Sport HE Active Fresh:

Water, Sodium alcoholethoxy sulfate, MEA citrate, linear alkylbenzenesulfonate, sodium salt, linear alkylbenzene sulfonate: MEA salt, alcoholethoxylate, sodium fatty acids, propylene glycol, diethylene glycol,polyethyleneimine ethoxylate propoxylate, diquaternium ethoxysulfate,Ethanol, sodium cumene sulfonate, borax, fragrance, DTPA, Sodiumbisulfate, disodium diaminostilbene disulfonate, Mannanase, cellulase,amylase, sodium formate, calcium formate, Lauramine oxide, Liquitint™Blue, Dimethicone/polydimethyl silicone.

Tide Plus Febreeze Freshness Spring & Renewal:

Water, sodium alcoholethoxy sulfate, linear alkyl benzene sulfonate:sodium/MEA salts, MEA citrate, propylene glycol, polyethyleneimineethoxylate, fragrance, ethanol, diethylene glycol, polyethyleneiminepropoxyethoxylate, protease, alcohol sulfate, borax, sodium fatty acids,DTPA, disodium diaminostilbene disulfonate, MEA, mannanase, gluconase,sodium formate, dimethicone, Liquitint™ Blue, tetramine.

Liquid Tide Plus with Febreeze Freshness, Sport HE Victory Fresh:

Water, Sodium alcoholethoxy sulfate, MEA citrate, linear alkylbenzenesulfonate, sodium salt, linear alkylbenzene sulfonate: MEA salt, alcoholethoxylate, sodium fatty acids, propylene glycol, diethylene glycol,polyethyleneimine ethoxylate propoxylate, diquaternium ethoxysulfate,ethanol, sodium cumene sulfonate, borax, fragrance, DTPA, Sodiumbisulfate, disodium diaminostilbene disulfonate, Mannanase, cellulase,amylase, sodium formate, calcium formate, Lauramine oxide, Liquitint™Blue, Dimethicone/polydimethyl silicone.

Tide Vivid White+Bright Powder, Original:

Sodium Carbonate, Sodium Aluminosilicate, Sodium Sulfate, LinearAlkylbenzene Sulfonate, Sodium Percarbonate,Nonanoyloxybenzenesulfonate, Alkyl Sulfate, Water, Silicate, SodiumPolyacrylate Ethoxylate, Polyethylene Glycol 4000, Fragrance, DTPA,Palmitic Acid, Protease, Disodium Diaminostilbene Disulfonate, Silicone,FD&C Blue 1, Cellulase, Alkyl Ether Sulfate.

Wash Assays

Mini Launder-O-Meter (MiniLOM) Model Wash System

MiniLOM is a mini wash system in which washes are performed in 50 mltest tubes placed in a Stuart rotator. Each tube simulates one smallwashing machine and during an experiment, each will contain a solutionof a specific detergent/enzyme system to be tested along with the soiledand unsoiled fabrics it is tested on. Mechanical stress is achieved viarotation (typically 20 rpm), and the temperature is controlled byplacement of the rotator in a heating cabinet/room.

Terg-O-toMeter (TOM) Wash Assay

The Terg-O-toMeter (TOM) is a medium scale model wash system that can beapplied to test 12 different wash conditions simultaneously. A TOM isbasically a large temperature controlled water bath with up to 12 openmetal beakers submerged into it. Each beaker constitutes one small toploader style washing machine and during an experiment, each of them willcontain a solution of a specific detergent/enzyme system and the soiledand unsoiled fabrics its performance is tested on. Mechanical stress isachieved by a rotating stirring arm, which stirs the liquid within eachbeaker. Because the TOM beakers have no lid, it is possible to withdrawsamples during a TOM experiment and assay for information on-line duringwash.

The TOM model wash system is mainly used in medium scale testing ofdetergents and enzymes at US or LA/AP wash conditions. In a TOMexperiment, factors such as the ballast to soil ratio and the fabric towash liquor ratio can be varied. Therefore, the TOM provides the linkbetween small scale experiments, such as AMSA and mini-wash, and themore time consuming full scale experiments in top loader washingmachines. Equipment: The water bath with 12 steel beakers and 1 rotatingarm per beaker with capacity of 500 or 1200 mL of detergent solution.Temperature ranges from 5 to 80° C. The water bath must be filled upwith deionised water. Rotational speed can be set up to 70 to 120rpm/min. Set temperature in the Terg-O-toMeter and start the rotation inthe water bath. Wait for the temperature to adjust (tolerance is +/−0.5°C.). All beakers shall be clean and without traces of prior testmaterial. The wash solution with desired amount of detergent,temperature and water hardness is prepared in a bucket. The detergent isallowed to dissolve during magnet stirring for 10 min. Wash solutionshall be used within 30 to 60 min after preparation. 800 ml washsolution is added into a TOM beaker. The wash solution is agitated at120 rpm and optionally one or more enzymes are added to the beaker. Theswatches are sprinkled into the beaker and then the ballast load. Timemeasurement starts when the swatches and ballast are added to thebeaker. The swatches are washed for 20 minutes after which agitation isterminated. The wash load is subsequently transferred from the TOMbeaker to a sieve and rinse with cold tap water. The soiled swatches areseparated from the ballast load. The soil swatches are transferred to a5L beaker with cold tap water under running water for 5 minutes. Theballast load is kept separately for the coming inactivation. The wateris gently pressed out of the swatches by hand and placed on a traycovered with a paper. Another paper is placed on top of the swatches.The swatches are allowed to dry overnight before subjecting the swatchesto analysis, such as measuring the color intensity using a Color Eye asdescribed herein.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

Assays

Assay 1: Hexosaminidase Activity

The hexosaminidase activity of the mature polypeptides with SEQ ID NO 7,SEQ ID NO 8, and SEQ ID NO 9, was determined using 4-nitrophenylN-acetyl-β-D-glucosaminide (Sigma-Aldrich) as substrate. The enzymaticreaction was performed in triplicates in a 96 well flat bottompolystyrene microtiter plate (Thermo Scientific) with the followingconditions: 50 mM 2-(N-morpholino) ethanesulfonic acid pH 6 buffer, 1.5mg/ml 4-nitrophenyl N-acetyl-β-D-glucosaminide and 20 μg/ml purifiedenzyme sample in a total reaction volume of 100 μl. Blank sampleswithout enzyme were run in parallel. The reactions were carried out at37° C. in a Thermomixer comfort (Eppendorf). After 10 minutes ofincubation, 5 μl 1 M NaOH was added to each reaction mixture to stop theenzymatic reaction. The absorbance was read at 405 nm using a POLARstarOmega plate reader (BMG LABTECH) to estimate the formation of4-nitrophenolate ion released because of enzymatic hydrolysis of the4-nitrophenyl N-acetyl-β-D-glucosaminide substrate. The results aresummarized in the table below. The table shows the average absorbancemeasured at 405 nm for each reaction performed in triplicates. It isseen that the absorbance is higher for the reaction carried out with SEQID NO 7, SEQ ID NO 8, SEQ ID NO 9, compared to blank without enzymewhich demonstrates that SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 exhibithexosaminidase activity.

TABLE 2 Hexosaminidase activity of SEQ ID NO 7, SEQ ID NO 8 and SEQ IDNO 9. ΔA405 nm Enzyme (A405 nm_(sample) − Enzyme concentration A405 nmA405 nm_(blank)) Blank  0 μg/ml 0.158 — SEQ ID NO 7 50 μg/ml 1.978 1.820SEQ ID NO 8 50 μg/ml 1.715 1.557 SEQ ID NO 9 50 μg/ml 2.455 2.297Assay 2: Hexosaminidase Activity

The hexosaminidase activity of the mature polypeptides with SEQ ID NO 7,SEQ ID NO 8, SEQ ID NO 9, and SEQ ID NO 10, was determined using4-Methylumbeliferyl N-acetyl-β-D-glucosaminide (Sigma-Aldrich) assubstrate. The enzymatic reaction was performed in triplicates in a 96well flat bottom polystyrene microtiter plate (Thermo Scientific) withthe following conditions: 20 mM 3-morpholinopropane-1-sulfonic acid pH 7buffer, 5 mM 4-Methylumbeliferyl N-acetyl-β-D-glucosaminide and 20 nMpurified enzyme sample in a total reaction volume of 200 μl. Blanksamples without enzyme were run in parallel. The reactions were carriedout at ambient temperature 20-25° C. The reaction kinetics was followedimmediately after mixing of enzyme and substrate using a SpectraMax M2eplate reader. Excitation wavelength was set to 368 nm and fluorescenceemission reading was done at 448 nm. The reaction was followed for 30min with 60 second intervals. Increase in fluorescence signal was usedto estimate the formation of 4-Methylumbeliferyl ion released because ofenzymatic hydrolysis of the 4-MethylumbeliferylN-acetyl-β-D-glucosaminide substrate. The results are summarized in thetable below. The table shows the average initial rate of reactionmeasured as relative fluorescence units per minute (RFU/min) usingexcitation at 368 nm and fluorescence emission at 448 nm for eachreaction performed in triplicates. It is seen that the reaction initialrate is higher for the reaction carried out with SEQ ID NO 7, SEQ ID NO8, SEQ ID NO 9, and SEQ ID NO 10 compared to blank without enzyme whichdemonstrates that SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, and SEQ ID NO10 exhibit hexosaminidase activity.

TABLE 3 Hexosaminidase activity of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO9, and SEQ ID NO 10. Δ Reaction initial rate (RFU/min) = (Reactioninitial rate_(sample) − Reaction initial rate_(blank)) Reaction ΔReaction Enzyme initial rate initial rate Enzyme concentration (RFU/min)(RFU/min) Blank  0 nM 0.6 — SEQ ID NO 7 20 nM 18.5 17.9 SEQ ID NO 8 20nM 10.1 9.5 SEQ ID NO 9 20 nM 13.1 12.5 SEQ ID NO 10 20 nM 82.6 82.1

EXAMPLES Example 1 Expression and Cloning of Hexosaminidases

The DNA encoding the hexosaminidase having the polypeptide comprised inSEQ ID NO 2 was isolated from a Terribacillus saccharophilus strainobtained from ATCC in 1969 (ATCC12327). The DNA encoding thehexosaminidase having the polypeptide comprised in SEQ ID NOS 4 and 6were isolated from a Terribacillus goriensis and a Terribacillussaccharophilus bacterial strains respectively, isolated from anenvironmental soil sample collected in USA. The DNA encoding thehexosaminidase having the polypeptide comprised in SEQ ID NOS 13 and 15were isolated later from two Terribacillus saccharophilus strains,isolated from environmental samples collected in USA. Chromosomal DNAfrom the four Terribacillus saccharophilus strains and the Terribacillusgoriensis strain was isolated by QIAamp DNA Blood Mini Kit (Qiagen,Hilden, Germany) and subjected to full genome sequencing using Illuminatechnology. The genome sequence was analyzed for protein sequences thathave glycosyl hydrolase domains (GH20, www.cazy.org). Three GH20 genesand corresponding sequences SEQ ID NOS: 1, 3, 5, 12 and 14 wereidentified from Terribacillus saccharophilus and Terribacillus goriensisstrains. The codon optimized synthetic DNA encoding the mature peptidesequences of the hexosaminidases were ordered from the company Geneart.The mature polypeptides are shown in SEQ ID NO 7, 8, 9 10 and 11.

TABLE 4 enzyme Donor SEQ ID NO 7 Terribacillus saccharophilus SEQ ID NO8 Terribacillus goriensis SEQ ID NO 9 Terribacillus saccharophilus SEQID NO 10 Terribacillus saccharophilus SEQ ID NO 11 Terribacillussaccharophilus

Example 2: Cloning and Expression of the Hexosaminidases

The codon optimized synthetic genes encoding the mature peptidesequences of the hexosaminidases or the polypeptides havinghexosaminidase activity is shown in SEQ ID NO 7, 8, 9, 10 and 11. Thesequences were inserted into a Bacillus expression vector as describedin WO 12/025577. Briefly, the DNA encoding the mature peptide of thehexosaminidase gene was cloned in frame to a Bacillus clausii secretionsignal (BcSP; with the following amino acid sequence:MKKPLGKIVASTALLISVAFSSSIASA (SEQ ID NO 16). BcSP replaced the nativesecretion signal in the gene. Downstream of the BcSP sequence, anaffinity tag sequence was introduced to ease the purification process(His-tag; with the following amino acid sequence: HHHHHHPR (SEQ ID NO17) The gene that was expressed therefore comprised the BcSP sequencefollowed by the His-tag sequence followed by the mature wild type GH20sequence i.e. the polypeptides with SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO9, SEQ ID NO 10 and SEQ ID NO 11. The final expression plasmid(BcSP-His-tag-GH20) was transformed into a Bacillus subtilis expressionhost. The GH20 BcSP-fusion gene was integrated by homologousrecombination into the Bacillus subtilis host cell genome upontransformation.

The gene construct was expressed under the control of a triple promotersystem (as described in WO 99/43835). The gene coding forchloramphenicol acetyltransferase was used as maker (as described in(Diderichsen et al., 1993, Plasmid 30: 312-315)). Transformants wereselected on LB media agar supplemented with 6 micrograms ofchloramphenicol per ml. One recombinant Bacillus subtilis clonecontaining the GH20 expression construct was selected and was cultivatedon a rotary shaking table in 500 ml baffled Erlenmeyer flasks eachcontaining 100 ml yeast extract-based media. After 3-5 days' cultivationtime at 30° C. to 37° C., the enzyme containing supernatant washarvested by centrifugation and the enzymes was purified by His-tagpurification.

Example 3: His Tag Purification Method

The His-tagged GH20 hexosaminidase enzymes were purified by immobilizedmetal chromatography (IMAC) using Ni²⁺as the metal ion on 5 mL HisTrapExcel columns (GE Healthcare Life Sciences). The purification took placeat pH 7 and the bound proteins were eluted with imidazole. The purity ofthe purified enzyme was checked by SDS-PAGE and the concentration of theenzyme determined by Absorbance 280 nm after a buffer exchange in 50 mMHEPES, 100 mM NaCl pH7.0

Example 4: Biofilm Assay

Staphylococcus aureus was kindly provided by Iñigo Lasa (Valle et al.,Mol Microbiol. 2003 May; 48 (4):1075-87). The strain was grown ontrypticase soy agar (TSA) at 37° C. overnight. Next day, a single colonywas transferred to 15 ml tripticase soy broth (TSB) and incubated 5hours at 37° C. under shaking. The culture was diluted 1:100 in TSB+1%glucose and 100 μL of the bacterial suspension was transferred to eachwell of a 96-well microtiter plates (Thermo Scientific, Nunclon DeltaSurface, cat #167008) and incubated 24 hours at 37° C. without shaking.Supernatant was aspirated and wells were washed with 100 μL of 0.9%sodium chloride and filled with 100 μL of either hard water or 3.3 gr/LModel detergent A containing 0 (control) or 20, 10, 5, 2.5, 1.25, 0.62,0.31, 0.16, 0.08, 0.04, 0.02 and 0.01 μg/mL of enzyme (the maturepolypeptides having SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10and SEQ ID NO 11). After incubation at 37° C. for 1 hour, wells werewashed with water and stained for 15 min with 100 μL of 0.095% crystalviolet solution (SIGMA V5265). Wells were then rinsed twice with 100 μLwater, dried and the plates were scanned.

The lowest concentration of each enzyme that could reduce the visibleformation of S. aureus biofilm after 1 hour incubation, in the presenceand absence of detergent was determined (see Table 5). All enzymes wereassayed per duplicate with similar results.

TABLE 5 Minimal concentration of enzyme that can reduce the visibleformation of S. aureus after 1 hour incubation in either hard water orModel detergent A. Minimal concentration Minimal concentration forbiofilm reduction for biofilm reduction in Model A in Hard water Enzymeμg/mL μg/mL SEQ ID NO 7 2.5 0.08 SEQ ID NO 8 2.5 0.16 SEQ ID NO 9 2.50.16 SEQ ID NO 10 0.31 <0.01 SEQ ID NO 11 10 0.02

Example 5: Deep-cleaning of Hexosaminidases in Liquid Model Detergent

Staphylococcus aureus 15981 (kind gift from Iñigo Lasa (Valle et al.,Mol Microbiol. 2003 May; 48 (4):1075-87) was used as model microorganismin the present example. S. aureus was restreaked on Tryptone Soya Agar(TSA) (pH 7.3) (CM0131; Oxoid Ltd, Basingstoke, UK) and incubated for 1day at 37° C. A single colony was inoculated into 10 mL of TSB and theculture was incubated for 16 hours at 37° C. with shaking (200 rpm).After propagation, the S. aureus culture was diluted (1:100) in freshTSB+1% glucose (24563; Roquette Freres) and 2 mL aliquots were added tothe wells of 12-well polystyrene flat-bottom microplates (3512; Costar,Corning Incorporated, Corning, N.Y., USA), in which round swatches(diameter 2 cm) of sterile polyester (WFK30A) had been placed. SterileTSB+1% glucose was added to control wells. After 48 h at 37° C. (staticincubation), the swatches were rinsed twice with 15° dH water. Fiverinsed swatches (sterile or with S. aureus) were placed in 50 mL testtubes and 10 mL of wash liquor (15° dH water with 0.2 g/L iron(III)oxide nanopowder (544884; Sigma-Aldrich) with 3.33 g/L liquid model Adetergent) and 0.2 ppm enzyme (mature polypeptide with SEQ ID NO 7, 8and 9) were added to each tube. Washes without enzyme were included ascontrols. The test tubes were placed in a Stuart rotator and incubatedfor 1 hour at 37° C. at 20 rpm. The wash liquor was then removed, andthe swatches were rinsed twice with 15° dH water and dried on filterpaper over night.

The color difference (L) values were measured using a Handheld MinoltaCR-300, and are displayed in table 6. Delta values(L_((swatch washed with enzyme))−L_((swatch washed without enzyme))) arealso indicated.

TABLE 6 Deep-cleaning effects of hexosaminidases in model detergent AEnzyme ΔL (Lwith enzyme − Enzyme concentration (ppm) L values Lwithoutenzyme) No enzyme 0 102.9 SEQ ID NO 7 0.2 115.1 12.3 SEQ ID NO 8 0.2115.5 12.7 SEQ ID NO 9 0.2 115.2 12.3

The results show that the hexosaminidases display deep-cleaningproperties in model detergent A.

Example 6: Deep-Cleaning Effects of Hexosaminidases in Liquid ModelDetergent

Staphylococcus aureus biofilms were grown on textile swatches (wfk30A)as described in example 5. Swatches incubated with sterile medium wereincluded as controls. For testing the deep-cleaning properties of thehexosaminidases, five rinsed swatches (sterile or with S. aureus) wereplaced in 50 mL conical centrifuge tubes and 10 mL of wash liquor (15°dH water with 0.2 g/L iron(III) oxide nanopowder (544884; Sigma-Aldrich)with 3.33 g/L liquid model A detergent) and 0.2 ppm (or 2 ppm) enzymewas added to each tube. No enzymes were added to the controls. The testtubes were placed on a Stuart rotator and incubated for 1 hour at 37° C.at 20 rpm. The wash liquor was then removed, and the swatches wererinsed twice with 15° dH water and dried on filter paper over night.

The color difference (L) values were measured using a Handheld MinoltaCR-300, and the relative wash efficiencies were calculated((L_(With enzyme)−L_(No enzyme))/(L_(Sterile swatch)−L_(No enzyme))*100)and are indicated in table 7.

TABLE 7 Percentage wash efficiency in liquid model detergent on S.aureus biofilm swatches Enzyme concentration Swatch Enzyme (ppm) % Washefficiency S. aureus biofilm 0 0.0 S. aureus biofilm SEQ ID NO 9 0.283.7 S. aureus biofilm SEQ ID NO 10 0.2 82.1 S. aureus biofilm SEQ ID NO11 0.2 62.5 S. aureus biofilm SEQ ID NO 11 2 89.1

Combined with the previous examples, the results show that all thepolypeptides of the invention have deep cleaning properties whencompared to samples comprising no enzyme.

Example 7: Deep-Cleaning Effects of Hexosaminidases on Pseudomonasfluorescens Biofilm Swatches

A Pseudomonas fluorescens isolate from Iceland, was used as modelmicroorganism in the present example. Pseudomonas fluorescens wasrestreaked on Tryptone Soya Agar (TSA) (pH 7.3) (CM0131; Oxoid Ltd,Basingstoke, UK) and incubated for 3 days at ambient temperature. Asingle colony was inoculated into 10 mL of TSB and the culture wasincubated for 16 hours at ambient temperature with shaking (Tetramax1000, 460 rpm). After propagation, the culture was diluted (1:100) infresh TSB and 1.65 mL aliquots were added to the wells of 12-wellpolystyrene flat-bottom microplates (3512; Costar, Corning Incorporated,Corning, N.Y., USA), in which round swatches (diameter 2 cm) of steriletextile (WFK20A) had been placed. Sterile TSB was added to controlwells. After 48 h incubation at ambient temperature (statically), theswatches were rinsed twice with 0.9% (w/v) NaCl. Five rinsed swatches(sterile or with P. fluourescens biofilm) were placed in 50 mL testtubes and 10 mL of wash liquor (15° dH water with 0.2 g/L iron(III)oxide nanopowder (544884; Sigma-Aldrich) with 3.33 g/L liquid model Adetergent) and 0.2 ppm or 2 ppm enzyme(s) was added to each tube. Washeswithout enzyme were included as controls. The test tubes were placed ina Stuart rotator and incubated for 1 hour at 30° C. at 20 rpm. The washliquor was then removed, and the swatches were rinsed twice with 15° dHwater and dried on filter paper over night.

The tristimulus light intensity (Y) values were measured using a DigiEYEcolour measurement and imaging system (VeriVide) equipped with a NikonD90 digital camera, and are displayed in table 8. Delta values(Y_((swatches washed with enzyme))−Y_((swatches washed without enzyme)))are also indicated.

TABLE 8 Deep-cleaning effects of the hexosaminidases on P. fluorescensbiofilms on textile Enzyme concentration Average Y Swatch Enzyme (ppm)values ΔY P. fluorescens biofilm 0 58.3 P. fluorescens biofilm SEQ ID NO7 0.2 66.1 7.8 P. fluorescens biofilm SEQ ID NO 7 2 66.2 7.8 P.fluorescens biofilm SEQ ID NO 8 0.2 66.7 8.4 P. fluorescens biofilm SEQID NO 8 2 66.7 8.4 P. fluorescens biofilm SEQ ID NO 9 0.2 67.0 8.6 P.fluorescens biofilm SEQ ID NO 9 2 67.2 8.9 P. fluorescens biofilm SEQ IDNO 10 0.2 67.7 9.4 P. fluorescens biofilm SEQ ID NO 10 2 67.6 9.3 P.fluorescens biofilm SEQ ID NO 11 0.2 63.0 4.6 P. fluorescens biofilm SEQID NO 11 2 66.9 8.6

Example 8 Deep-cleaning Effects of Hexosaminidases in Liquid ModelDetergent Against EPS from Different Microorganisms

Crude extracts of biofilm extracellular polymeric substances (EPS) wereprepared from Staphylococcus aureus 15981 (kind gift from Iñigo Lasa(Valle, J., A. Toledo-Arana, C. Berasain, J. M. Ghigo, B. Amorena, J. R.Penades, and I. Lasa. 2003, Mol. Microbiol. 48:1075-1087),Staphylococcus cohnii (textile isolate, Denmark, 0437F4), Pseudomonasfluorescens (Isolate from Iceland) and Acinetobacter iwoffi (textileisolate from Denmark. For S. aureus, the extract was made as follows:The strain was restreaked on Tryptone Soya Agar (TSA) (pH 7.3) (CM0131;Oxoid Ltd, Basingstoke, UK) and incubated for 3 days at 37° C. 500 mL ofTSB+1% glucose (24563; Roquette Freres) was then inoculated, aliquotedinto 50 ml conical centrifuge tubes (339652; Thermo Scientific Nunc) (33ml in each), and incubated for 24 hours at 37° C. with shaking (200rpm). The cells were subsequently pelleted by centrifugation (10 min,6000 g, 25° C.), pooled and resuspended in a total of 5 ml 3M NaCl. Thesuspension was vortexed vigorously and incubated for 15 min at ambienttemperature to extract the surface-associated EPS. The cells were thenre-pelleted (10 min, 5000 g, 25° C.) and the EPS-containing supernatantwas retrieved. The supernatant was sterile filtered twice (0.45 μmfollowed by 0.2 μm), tested for sterility and stored at −20° C. untilfurther use.

The S. cohnii isolate was restreaked on TSA, incubated for 3 days at 37°C., and used to inoculate an overnight culture (10 ml TSB+1% glucose,incubated at 37° C., 200 rpm). This culture was then diluted (1:100)into 250 ml fresh medium (TSB+1% glucose) in a Corning® CelIBIND® 225cm² Angled Neck Cell Culture Flasks with Vent Cap (Product #3293), andthe flask was incubated at 37° C. for 2 days. The biofilm culture waspelleted by centrifugation (10 min, 8000 g, 25° C.), resuspended in 2.5ml 3M NaCl and incubated for 30 min at room temperature. The cells werethen re-pelleted (10 min, 5000 g, 25° C.), and the EPS-containingsupernatant was retrieved, sterile filtered (0.20 μm, 16534-K, Minisart,Sartorius Stedim) and stored at −20° C. until use.

P. fluorescens was restreaked on TSA and incubated for 1 day at 20° C.The strain was inoculated into 10 mL of TSB and the culture wasincubated statically for 16 hours at 20° C. After propagation, theculture was diluted (1:100) in 400 ml M63 supplemented medium (15 mM(NH₄)₂SO₄, 100 mM KH₂PO₄, 1.8 μM FeSO₄, 1 mM MgSO₄.7H2O, 0.4% (w/v)glycerol, 0.2% (w/v) Casamino acids and 0.0001% (w/v) Thiamine), addedto a Corning® CelIBIND® 225 cm² Angled Neck Cell Culture Flasks withVent Cap and incubated statically for 3 days at 20° C. The biofilmculture was subsequently pelleted by centrifugation (10 min, 8000 g, 25°C.), and the cells resuspended in 4 ml 3M NaCl and incubated for 30 minat 30° C. to extract the surface-associated EPS. The EPS-containingsupernatant obtained after centrifugation (10 min, 5000 g, 25° C.) wasthen sterile filtered and stored at −20° C. until further.

A crude EPS extract from A. iwoffi was prepared as follows: The strainwas restreaked on a TSA plate and incubated for 3 days at 30° C. 10 mlLB medium (L3152, Fluka) was then inoculated with a single colony, andincubated at 30° C., 200 rpm for 48 h. The culture was diluted (1:100)in 300 ml fresh LB, added to Corning® CelIBIND® 225 cm² Angled Neck CellCulture Flasks with Vent Cap and incubated statically for 4 days at 30°C. The biofilm culture was subsequently pelleted by centrifugation (10min, 8000 g, 25° C.), and the pellet was resuspended in a total of 3 ml3M NaCl and incubated for 30 min at room temperature. The EPS-containingsupernatant obtained after centrifugation (10 min, 5000 g, 25° C.) wassterile filtered (0.20 μm, 16534-K, Minisart, Sartorius Stedim) andstored at −20° C. until use.

For wash performance testing, 50 ul aliquots of the different crude EPSextracts were spotted on sterile textile swatches (WFK20A) and incubatedfor 15 min at ambient temperature. The swatches (sterile or with EPS)were placed in 50 mL test tubes and 10 mL of wash liquor (15° dH waterwith 0.2 g/L iron(III) oxide nano-powder (544884; Sigma-Aldrich) with3.33 g/L liquid model A detergent) and 0.2 μg/ml or 2 μg/ml enzyme wasadded to each tube. Washes without enzyme were included as controls. Thetest tubes were placed in a Stuart rotator and incubated for 1 hour at37° C. at 20 rpm. The wash liquor was then removed, and the swatcheswere rinsed twice with 15° dH water and dried on filter paper overnight. The tristimulus light intensity (Y) values were measured using aDigiEYE colour measurement and imaging system (VeriVide) equipped with aNikon D90 digital camera, and are displayed in table 9.

Delta values(Y_((swatches washed with enzyme))−Y_((swatches washed without enzyme)))and relative wash efficiency values((L_(With enzyme)−L_(No enzyme))/(L_(Sterile swatch)−L_(No enzyme))*100))are also indicated.

TABLE 9 Deep-cleaning effects of the hexosaminidase shown in SEQ ID NO 9in liquid model detergent on EPS from different microorganisms EnzymeWash concentration Average Y efficiency Origin of EPS (ppm) values ΔY(%) Clean textile, no EPS 0 85.4 S. aureus 0 41.7 S. aureus 0.2 82.741.1 93.8 S. aureus 2 84.3 42.6 97.4 S. cohnii 0 73.3 S. cohnii 0.2 84.611.3 93.0 S. cohnii 2 84.7 11.4 93.8 P. fluorescens 0 54.4 P.fluorescens 0.2 86.0 31.6 101.8 P. fluorescens 2 85.5 31.1 100.1 A.Iwoffii 0 76.3 A. Iwoffii 0.2 84.9 8.6 94.0 A. Iwoffii 2 84.7 8.4 91.8

The data clearly shows that the hexosaminidases show deep-cleaningproperties against biofilm EPS from various microorganisms, includingboth gram-positive and gram-negative bacteria.

Example 9: Malodor Reduction of Hexosaminidase in Liquid Model Detergent

EPS were purified as described in e.g. example 8. After purification, 50μL aliquots of EPS were added to the wells of 12-well polystyreneflat-bottom microplates (3512; Costar, Corning Incorporated, Corning,N.Y., USA), in which round swatches (diameter 2 cm) of sterile polyester(WFK30A) had been placed. The swatches were incubated for 15 min (staticincubation) before proceeding. Six swatches were placed in 50 mL testtubes and 10 mL of wash liquor (15° dH water with 3.33 g/L liquid modelA detergent) and 0.2 ppm, 2.0 ppm or 20.0 ppm enzyme (SEQ ID NO 9) wereadded to each tube. Washes without enzyme were included as controls. Thetest tubes were placed in a Stuart rotator and incubated for 1 hour at37° C. at 20 rpm. The wash liquor was then removed, and the swatcheswere rinsed twice with 20 mL 15° dH water and dried on filter paper overnight.

Odor Chamber Setup and Analysis of Volatiles on Textile Using anElectronic Nose

To test for the binding of gaseous volatiles, all dried swatches wererandomly distributed in a square plastic box (30.7 cm×21.9 cm×6.0cm—inside measurements). In a 10 mL glass beaker position in the center,5 mL of a mixture of various aldehydes (Pentanal 160 mM, Hexanal 40 mM,Heptanal 80 mM, (E)-2-Heptenal 20 mM, Octanal 30 mM, Nonanal 10 mM,Decanal 8 mM, (E)-2-Decenal 10 mM and (E,E)-2-Decadienal 12 mM—allchemical purchased from Sigma-Aldrich) were added. The odor chamber wasclosed with a lid and wrapped in Parafilm®. The swatches were incubatedat room temperature for 16 h, before individually being placed in thebottom of 20 mL GC-MS vials (Mikrolab Aarhus NS, Aarhus, Denmark) andcapped with silicone screw top lids (Mikrolab Aarhus NS, Aarhus,Denmark). From each sample 5 mL headspace was analyzed in a:

-   -   Heracles II Electronic nose from Alpha M.O.S., France (double        column gas chromatograph with 2 FIDs, column 1: Restek MXT-5 10        m×0.18 mm×0.2 μm and column 2: Restek MXT-1701 10 m×0.18 mm×0.2        μm) after 20 minutes incubation at 40° C. Samples were run in a        randomized order.

TABLE 10 Malodor removal by hexosaminidase in Model A model detergent.Odor reduction 0.2 ppm Without with 0.2 ppm hexosaminidase enzymehexosaminidase Volatile (SEQ ID NO 9) (Relative (SEQ ID NO 9) compound(Relative intensity) intensity) Reduction in % Pentanal 917323 112885319 Hexanal 207470 280177 26 Heptanal 398361 621542 33 (E)-2-Heptenal59477 91362 35 Octanal 103164 172130 37 Nonanal 88695 130790 32 Decanal26768 44650 40 (E)-2-Decenal 18097 36608 51 (E,E)-2- 49063 101721 52Decadienal

The results show that hexosaminidase display malodor reductionproperties in model detergent A.

Example 10: Deep-Cleaning of Hexosaminidases in Different Detergents

A crude EPS (extracellular polymeric substances) extract was preparedfrom Pseudomonas fluorescens (Isolate from Iceland) as described inexample 8. For wash performance testing, 50 ul aliquots of this extractwere spotted on sterile textile swatches (WFK20A) and left to soak for15 min at ambient temperature. The swatches (sterile or with EPS) wereplaced in 50 mL conical centrifuge tubes and 10 mL of wash liquor (15°dH water with 0.2 g/L iron(III) oxide nano-powder (544884;Sigma-Aldrich) and with detergent (3.33 g/L liquid model A detergent,2.0 g/L model N or 4.6 g/L Persil Universal Gel®) was added. The enzymewas added to a final concentration of 0.2 μg/ml. Tubes without enzymeswere included as controls. The test tubes were placed in a Stuartrotator and incubated for 1 hour at 37° C. at 20 rpm. The wash liquorwas then removed, and the swatches were rinsed twice with 15° dH waterand dried on filter paper over night. The tristimulus light intensity(Y) values were measured using a DigiEYE colour measurement and imagingsystem (VeriVide) equipped with a Nikon D90 digital camera, and aredisplayed in table 11.

Delta values(Y_((swatch washed with enzyme))−Y_((swatch washed without enzyme))) arealso indicated.

TABLE 11 Deep-cleaning of hexosaminidases in different detergents on P.fluorescens EPS swatches Persil Universal Gel Enzyme conc. Model A ModelN (commercial detergent) Swatch Enzyme (ppm) Y values ΔY Y values ΔY Yvalues ΔY No EPS — 0 85.6 61.7 84.4 EPS — 0 49.7 51.9 55.2 EPS Seq IDNO. 7 0.2 85.1 35.5 74.1 22.2 82.8 27.6 EPS Seq ID NO. 8 0.2 83.5 33.875.3 23.4 83.5 28.3 EPS Seq ID NO. 9 0.2 85.0 35.3 73.4 21.5 81.8 26.5EPS Seq ID NO. 10 0.2 86.0 36.3 74.8 22.9 84.7 29.5 EPS Seq ID NO. 110.2 81.5 31.8 72.4 20.5 77.5 22.2

Example 11: Wash Performance in TOM

A crude EPS extract was prepared from Pseudomonas fluorescens (Isolatefrom Iceland,) as described in example 8. The extract was sterilefiltered prior to use, and 100 μl aliquots were spotted on textileswatches (wfk20A (Polyester/Cotton mix (65%/35%)), 50 mm×50 mm), andincubated at room temperature for 10 min. The Terg-o-tometer wash wasperformed at follows; 0.4 g/L iron(III) oxide nano-powder (544884;Sigma-Aldrich) and 3.33 g/L liquid model detergent A was weighed out andleft to soak for 1 h prior to the wash. The dirty detergent was thenadded to 1000 ml pre-warmed water hardness (15° dH) in the TOM beakers,and allowed to dissolve for 10 min (maximum agitation). The agitationlevel was then reduced to 90 rpm, and the textile swatches (two EPSswatches as well as ballast material (wfk10A, wfk30A) to reach a totalof 10 g textile/beaker) were added and washed with or without enzyme(0.2 ppm) for 35 min at 30° C. After wash, all swatches were rinsedtwice in tap water and dried on filter paper over night. The remisssion(REM^(460 nm)) values were measured using a Macbeth Color-Eye 7000(CE7000), and are displayed in table 12.

Delta values (REM^(460 nm) _((swatches washed with enzyme))−REM^(460 nm)_((swatches washed without enzyme))) are also indicated.

TABLE 12 Deep-cleaning properties of hexosamidases on EPS swatches,washed in TOM Enzyme Average concen- Remission tration (460 nm) ΔRem(460Swatch Enzyme (ppm) values nm) Clean textile, no EPS 60.4 P. fluorescensEPS SEQ ID NO 9 0 47.1 P. fluorescens EPS SEQ ID NO 9 0.2 65.3 18.2

Example 12: Wash Performance in Full-scale Wash

A crude EPS extract was prepared from Pseudomonas fluorescens (Isolatefrom Iceland) as described in example 8. The extract was spotted (200 μlaliquots) on a prewashed T-shirt (Anvil sustainable t-shirt (50%polyester/50% cotton)) that had been cut down the middle, and the spotswere left to dry at room temperature for 30 min prior to the wash. Thetwo halves of the T-shirt were washed independently in a Miele LaundryWashing Machine (Miele Softtronic, W2245) using tap water and 0.16 g/Liron(III) oxide nano-powder (544884; Sigma-Aldrich) in 3.33 g/L liquidmodel A detergent. The 40° C. Color program was used (run time 1 h, 26min). After wash, the two halves of the T-shirt were hang-dried at roomtemperature overnight. The remisssion (REM^(460 nm)) values weremeasured using a Macbeth Color-Eye 7000 (CE7000), and are displayed intable 13. Delta values (REM^(460 nm)_((swatches washed with enzyme))−REM^(460 nm)_((swatches washed without enzyme))) are also indicated.

TABLE 13 Deep-cleaning of hexosaminidases in full-scale washing machineEnzyme concen- Average EPS spotted on tration Rem^(460 nm) T-shirtEnzyme (ppm) values ΔRem^(460 nm) P. fluorescens EPS SEQ ID NO 9 0 22.4P. fluorescens EPS SEQ ID NO 9 0.2 45.7 23.3

Example 13: EPS Hydrolysis Measurements Using FluorescentWGA-Alexafluor488 Assay

The Pseudomonas fluorescens strain isolated from Iceland was used asmodel microorganisms in the present example. The Pseudomonas fluorescensstrain was restreaked on Tryptone Soya Agar (TSA) (pH 7.3) (CM0131;Oxoid Ltd, Basingstoke, UK) and incubated for 1 day at 23° C. The strainwas inoculated into 10 mL of TSB and the culture was incubated withshaking for 16 hours at 23° C. The overnight culture was diluted (1:100)in 200 ml M63 supplemented medium (15 mM (NH4)2SO4, 100 mM KH2PO4, 1.8μM FeSO4, 1 mM MgSO4.7H2O, 0.4% (w/v) glycerol, 0.2% (w/v) Casaminoacids and 0.0001% (w/v) Thiamine) added to a Corning® CelIBIND® 225 cm²Angled Neck Cell Culture Flask with Vent Cap (Product #3293) andincubated statically for 5 days at 23° C. Each biofilm culture wassubsequently transferred to four 50 ml falcon tubes (Corning #430820)and pelleted by centrifugation (10 min, 8000 g, 25° C.), and thesupernatants were discarded completely. The residual pellets from eachof the four falcons were resuspended in 0.450 ml 3M NaCl to extract thesurface-associated EPS (extracellular polymeric substances) and pooledin one test tube (5 ml, Eppendorf #0030119401). The suspension wascentrifuged at 5000 g for 10 min at 25° C. and the 1.8 ml supernatantwas transferred to a new test tube as EPS fraction and stored at −20° C.until further use (termed crude EPS). 150 ul aliquots of the crude EPSwere dispensed in three Eppendorf tubes and mixed with 150 ul of anenzyme solution containing 80 ppm of either SEQ ID NO 9 or 50 mMHepes100 mM NaCl buffer alone and incubated 1 hr at room temperature. 50ul aliquot samples were retrieved from each tube and then added to thewells of a nunc Maxisorp black microtiter plate (ThermoScientific#437111) in which round swatches of sterile prewashed swatches (wfk 20A,polyester/cotton 65%/35%) had been placed. The plate was incubated atroom temperature for 15 minutes. Supernatants were then removed fromeach well and swatches were washed with 100 ul water. Next, 50 ul ofWGA-Alexa fluor488 dye (10 ug/ml; excitation/emission maxima ˜495/519nm; Thermo Fischer Scientific, #W11261) was added to each well. AlexaFluor® 488 WGA binds to sialic acid and N-acetylglucosaminyl residues.The plate was incubated at room temperature for 15 min. Samples werefinally washed with 100 ul water and measured in the SpectraMax M3instrument. Each of the three samples corresponding to the crude EPSmixed with the enzyme solutions and buffer control, were tested on 8swatches placed in 8 wells. The measurements obtained including thestandard deviations are listed below in table 14.

TABLE 14 Fluorescent measurements with WGA-Alexa Fluor488 dye of crudeEPS from P. fluorescens strain treated with either buffer or SEQ ID NO9. sample average measurement Buffer control 416 SEQ ID NO 9 121

Results from the fluorescent measurements show that the EPS extract fromthe P. fluorescens strain is labelled with WGA-Alexa Fluor488 suggestingthat the EPS contains N-acetylglucosaminyl residues and is sensitive tohexosaminidase (SEQ ID NO 9) hydrolysis.

Example 14: Wheat-germ Agglutinin (WGA)-staining of Washed EPS Swatches

A crude EPS extract was prepared from Staphylococcus aureus 15981 (kindgift from Iñigo Lasa (Valle, J., A. Toledo-Arana, C. Berasain, J. M.Ghigo, B. Amorena, J. R. Penades, and I. Lasa. 2003, Mol. Microbiol.48:1075-1087) as described in example 8 with minor modifications: Theculture was incubated for 24 h at 37° C. in TSB+1% glucose prior to EPSextraction. For washing, 100 ul aliquots of the EPS were spotted onsterile textile swatches (prewashed wfk20A textile, 65% Polyester/35%Cotton) and incubated for 20 min at ambient temperature. The swatches(sterile or with EPS) were placed in 50 mL test tubes and 10 mL of washliquor (15° dH water with 3.33 g/L liquid model A detergent) and 20μg/ml enzyme was added to each tube. Washes without enzyme were includedas controls. The test tubes were placed in a Stuart rotator andincubated for 1 hour at 37° C. at 20 rpm. The wash liquor was thenremoved, and the swatches were rinsed twice with 15° dH water and placedin a 12-well polystyrene flat-bottom microplate (3512; Costar, CorningIncorporated, Corning, N.Y., USA). The swatches were then stained withAlexa Fluor® 488 Conjugated Wheat Germ Agglutinin (WGA) (10 μg/ml in1×PBS) for 30 min at room temperature, rinsed twice with 1×PBS andmeasured by well-scanning in a BMG CLARIOstar microplate reader(Excitation at 488 nm, emission at 525 nm). The fluorescence intensityvalues are shown in table 15.

The relative substrate removal compared to the control(FIU_(with enzyme)−FIU_(without enzyme)) is also shown.

TABLE 15 Wheat-germ agglutinin (WGA)-staining of EPS swatches washedwith or without hexosaminidase (SEQ ID NO 9). Fluorescent signal Enzymeintensity (488 nm(Ex), concentration 525 nm(Em), background % remainingSwatch (ppm) subtracted) substrate S. aureus EPS 0 49082 100 S. aureusEPS 20 3258 6.6

When WGA lectin has affinity for N-acetylglucosaminyl residues, theresults shows that the hexosaminidase treatment improves substrateremoval from the textile during the wash.

Example 15: Construction of Clades and Phylogenetic Trees

The Glyco_hydro_20 domain includes the polypeptides of the inventionhaving hexosaminidase e.g. PNAG activity and comprises the WND domain aswell as the clusters such as the clades. A phylogenetic tree wasconstructed, of polypeptide sequences containing a Glyco_hydro_20domain, as defined in PFAM (PF00728, Pfam version 31.0 Finn (2016).Nucleic Acids Research, Database Issue 44:D279-D285). The phylogenetictree was constructed from a multiple alignment of mature polypeptidesequences containing at least one Glyco_hydro_20 domain. The sequenceswere aligned using the MUSCLE algorithm version 3.8.31 (Edgar, 2004.Nucleic Acids Research 32(5): 1792-1797), and the trees were constructedusing FastTree version 2.1.8 (Price et al., 2010, PloS one 5(3)) andvisualized using iTOL (Letunic & Bork, 2007. Bioinformatics 23(1):127-128). The polypeptide sequences containing a Glyco_hydro_20 domaincomprises several motifs; one example is GXDE (SEQ ID NO 18), situatedin positions 158 to 161 in Terribacillus saccharophilus (SEQ ID NO 9).Residues D and E are the key catalytic residues of Glyco_hydro_20enzymes (position 160 to 161 in SEQ ID NO 9).

The polypeptides in Glyco_hydro_20 can be separated into multipledistinct sub-clusters, or clades as listed below. The distinct motifsfor each clade are described in details below.

Generation of IAS Domain

A domain, preferably shared by the polypeptides of the invention, wasidentified. This domain has not been described previously. The domain istermed IES and polypeptides of this domain comprises Glyco_hydro_20domain polypeptides of bacterial origin and are in addition to havingPNAG activity, characterized by comprising certain motifs. Thepolypeptides of the domain comprise the motif example[EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19), correspondingto ESYAIAS at position 44 to 50 of SEQ ID NO 9.

Generation of WND Domain

A domain, preferably shared by the polypeptides of the invention, wasidentified. This domain has not been described previously. The domain istermed WND and polypeptides of this domain comprise Glyco_hydro_20domain polypeptides of bacterial origin and are in addition to havingPNAG activity, characterized by comprising certain motifs. Thepolypeptides of the domain comprise the motif example[VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO 20), corresponding to pos 156 to163 of SEQ ID NO 9, where G and DE (corresponding to positions 158 and160-161 of SEQ ID NO 9) are fully conserved in DSP3 clade and part ofthe active site. Residues D and E are the key catalytic residues ofGlyco_hydro_20 enzymes (position 160 to 161 in SEQ ID NO 9). Anothermotif which may be comprised by the polypeptides of the WND domain isWND[SQR][IVL][TLVM] (SEQ ID NO 21), 193 to 198 in SEQ ID NO 9, where W(pos 193 in SEQ ID NO 9) is part of the active site pocket andputatively involved in binding of the N-acetyl group of the PNAGsubstrate.

Generation of QSTL Clade

The QSTL clade comprises WND domain polypeptides of bacterial origin,having hexosaminidase e.g. PNAG activity. The polypeptides of the cladecomprise the motif example QSTL (SEQ ID NO 22), corresponding to pos 216to 219 of SEQ ID NO 9, where all four amino are fully conserved in QSTLclade. The motif is part of a putative “lid” of the enzymes' TIM barrelstructure and putatively involved in substrate binding. Another motifwhich may be comprised by the polypeptides of the QSTL clade is NKFFY(SEQ ID NO 23), 273 to 277 in SEQ ID NO 9. A further motif which may becomprised by the polypeptides of the QSTL clade is NLD[DR]S (SEQ ID NO24), corresponding to amino acids 204 to 208 in SEQ ID NO 9.

An alignment of the polypeptides of the invention is shown in FIG. 2. Aphylogenetic tree of the polypeptides of the invention is shown in FIG.3.

The invention claimed is:
 1. A composition comprising (a) at least 0.001ppm of a polypeptide having hexosaminidase activity, wherein thepolypeptide is selected from the group consisting of (i) a polypeptidecomprising the amino acid sequence of SEQ ID NO: 7; (ii) a polypeptidehaving at least 97% sequence identity to SEQ ID NO: 8; (iii) apolypeptide having at least 97% sequence identity to the polypeptide ofSEQ ID NO: 9; (iv) a polypeptide having at least 80% sequence identityto the polypeptide of SEQ ID NO: 10; and (v) a polypeptide having atleast 85% sequence identity to the polypeptide of SEQ ID NO: 11; and (b)at least one adjunct ingredient selected from the group consisting of atleast one builder, at least one surfactant, and at least one bleachcomponent; wherein the composition is in the form of a liquid, gel,powder, granulate, paste, or spray composition.
 2. The composition ofclaim 1, wherein the polypeptide having hexosaminidase activitycomprises the amino acid sequence of SEQ ID NO:
 7. 3. The composition ofclaim 1, wherein the polypeptide having hexosaminidase activity has atleast 97% sequence identity to SEQ ID NO:
 8. 4. The composition of claim1, wherein the polypeptide having hexosaminidase activity comprises theamino acid sequence of SEQ ID NO:
 8. 5. The composition of claim 1,wherein the polypeptide having hexosaminidase activity has at least 97%sequence identity to the polypeptide of SEQ ID NO:
 9. 6. The compositionof claim 1, wherein the polypeptide having hexosaminidase activitycomprises the amino acid sequence of SEQ ID NO:
 9. 7. The composition ofclaim 1, wherein the polypeptide having hexosaminidase activity has atleast 95% sequence identity to the polypeptide of SEQ ID NO:
 10. 8. Thecomposition of claim 1, wherein the polypeptide having hexosaminidaseactivity comprises the amino acid sequence of SEQ ID NO:
 10. 9. Thecomposition of claim 1, wherein the polypeptide having hexosaminidaseactivity has at least 95% sequence identity to the polypeptide of SEQ IDNO:
 11. 10. The composition of claim 1, wherein the polypeptide havinghexosaminidase activity comprises the amino acid sequence of SEQ ID NO:11.
 11. The composition of claim 1, wherein the polypeptide hasN-acetylglucosaminidase activity and/or β-N-acetylglucosaminidaseactivity.
 12. The composition of claim 1, wherein the polypeptidecomprises one or more of the motif(s) GXDE (SEQ ID NO 18),[EQ][NRSHA][YVFL][AGSTC][IVLF][EAQYN][SN] (SEQ ID NO 19),[VIM][LIV]G[GAV]DE[VI][PSA] (SEQ ID NO: 20), WND[SQR][IVL][TLVM] (SEQ IDNO: 21), QSTL (SEQ ID NO 22), NKFFY (SEQ ID NO: 23), and NLD[DR]S (SEQID NO: 24).
 13. The composition of claim 1, wherein the polypeptidecomprises the amino acid sequence of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO9, SEQ ID NO 10, or SEQ ID NO
 11. 14. The composition of claim 1,wherein the composition is a cleaning composition.
 15. The compositionof claim 1, which comprises at least one builder in an amount from about5 wt. % to about 60 wt. % weight, wherein the builder is selected fromthe group consisting of phosphates, sodium citrate builders, sodiumcarbonate, sodium silicate, sodium and zeolites.
 16. The composition ofclaim 1, which comprises at least one bleaching component in an amountfrom about 1 wt. % to about 40 wt. %, wherein the bleach component is apercarbonate or bleach catalyst.
 17. The composition of claim 1, whichcomprises anionic surfactants and/or non-ionic surfactants in an amountfrom about 2 wt. % to about 60 wt. %.
 18. A method for laundering anitem, comprising: a) exposing the item to a wash liquor comprising acomposition of claim 1; and b) completing at least one wash cycle. 19.The method of claim 18, wherein the item is a textile.
 20. The method ofclaim 18, further comprising rinsing the item.